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QUANTITATIVE ANALYSIS AND HEALTH RISK
ASSESSMENT OF NOVEL BROMINATED FLAME
RETARDANTS IN HOUSE DUST
Taya Huang
MSc Thesis
Master's Degree Programme in Environmental Health Risk Assessment
University of Eastern Finland, Faculty of Science and Forestry
Department of Environmental and Biological Sciences
28 April, 2017
UNIVERSITY OF EASTERN FINLAND, Faculty of Science and Forestry
Department of Environmental Science
Master's Degree Programme in Environmental Health Risk Assessment
Taya Huang: Quantitative Analysis and Health Risk Assessment of Novel Brominated Flame
Retardants in House Dust
MSc thesis 66 pages, 8 appendixes ( 74 pages)
Supervisors: Panu Rantakokko, PhD; Matti Viluksela, PhD
28 April, 2017
________________________________________________________________________
Keywords: flame retardants, brominated flame retardants, method development,
environmental health risk assessment, indoor dust
ABSTRACT
This thesis discusses the method development and validation for analysis of flame retardants
in house dust that was performed between May-August 2015 based on the method developed
by Van den Eede et al. 2012. The principle aim of this method development is to develop and
validate an effective analysis method for indoor dust containing selected novel and emerging
brominated flame retardants (BFR), organophosphorous flame retardants (OPFR), as well as
polybrominated diphenyl ether (PBDE). In the second part of this thesis, a human health risk
assessment for one of the above selected BFRs, namely Bis(2-ethyl-1hexyl)
tetrabromophthalate (BEH-TEBP) is performed based on currently available scientific
literature and estimated exposure from indoor dust ingestion. For the validation, Limit of
Quantification (LOQ) & Measurement Uncertainties were acceptable for all BFRs. LOQ of
BFRs ranged between 0.5 – 5.0 ng/g. For OPFR, LOQ ranged between 6.9-613 ng/g.
Precision for OPFR was good, but accuracy was poor compared to other labs’ analysis result
for the certified material for indoor dust SRM 2585. Therefore, method for OPFR analysis
requires further testing that was not conducted within the scope of this thesis. For the purpose
of estimating exposure, unpublished results from the National Institute for Health and Welfare
(THL) were used. The detected amount of BEH-TEBP from children’s room in Kuopio,
Finland (n=40) had a median of 106.3 ng/g, with a range of 22.8 ng/g – 887.2 ng/g (5th to 95th
percentile). A Risk Characterisation Ratio (RCR) has been calculated based on the derived
no-effect level (DNEL) of 0.37 mg/kg bw/day for long-term oral exposure in the general
population, taken from the registration dossier of BEH-TEBP. All RCR derived for given
exposure scenarios are less than 1, meaning that the risk is adequately controlled. However, it
must be kept in mind that combined exposure has not been taken into consideration.
ACKNOWLEDGEMENTS
The laboratory work for this thesis was performed between May-August 2015. The method
development and validation for analysis of flame retardants in house dust described in this
thesis has been done according to the instructions and guidance from Dr. Panu Rantakokko of
the National Institute for Health and Welfare (THL), Kuopio. The official validation report for
the first validation has been written by Dr. Rantakokko for THL. The method development
was still on-going as of August 2016. This thesis includes only the part of the work I have
been involved in. This laboratory work forms the first part of my thesis. The second part of
my thesis focuses on the environmental health risk assessment of selected brominated flame
retardants.
ABBREVIATIONS AND DEFINITIONS
Terms
BFR Brominated Flame Retardant
DNEL Derived No-Effect Level
FR Flame Retardant
LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantification
NOAEL No Observed Adverse Effect Level
NOEL No Observed Effect Level
OPFR Organophosphorous Flame Retardant
RCR Risk Characterisation Ratio
REACH European Commission Regulation EC
1907/2006 concerning the Registration,
Evaluation, Authorisation and Restriction of
Chemicals
THL National Institute for Health and Welfare
USEPA Unites States Environmental Protection
Agency
Flame Retardants
ab-DBE-DBCH alpha/beta-Tetrabromoethylcyclohexane
BEH-TEBP Bis(2-ethyl-1hexyl)tetrabromophthalate
BTBPE 1,2-Bis(2,4,6-tribromophenoxy)ethane
DEHP di-2-ethyl hexyl phthalate
EHDPP 2-Ethylhexyl diphenyl phosphate
EH-TBB 2-Ethylhexyl-2,3,4,5-tetrabromobenzoate
PBDE Polybrominated diphenyl ether
PBT Pentabromotoluene
TBOEP Tris(2-butoxyethyl)phosphate
TBP-DBPE 2,3-Dibromopropyl 2,4,6-tribromophenyl ether
TCEP Tris(2-chloroethyl) phosphate
TCIPP Tris(1-chloropropan-2-yl) phosphate
TDBPP Tris(2,3-dibromopropyl) phosphate
TDCIPP Tris(1,3-dichloroisopropyl) phosphate
TEHP Tris(2-ethylhexyl)phosphate
TIBP Tri(isobutyl)phosphate
TMPP Tris (methylphenyl) phosphate (isomer mixture)
TNBP Tri-n-butylphosphate
TPHP Triphenyl phosphate
Chemicals
DCM Dichloromethane
HEX Hexane
Contents
1. INTRODUCTION 6
2 ANALYTICAL METHOD DEVELOPMENT FOR BFRs IN HOUSE DUST 12
2.1 MATERIALS AND METHODS 12
2.1.1 CHEMICALS AND INSTRUMENTS 12
2.1.2 GC-HRMS CONDITIONS 13
2.1.3 FRACTIONATION AND CLEAN-UP 14
2.1.4 EXTRACTION 20
2.1.5 VALIDATION 20
2.2 RESULTS AND DISCUSSION 26
2.2.1 FRACTIONATION AND CLEAN-UP OPTIMIZATION 26
2.2.2 EXTRACTION 27
2.2.3 SUMMARY OF SAMPLE TREATMENT IN FLOW CHART 29
2.2.4 RESULTS OF VALIDATION 29
2.2.5 CONCLUSIONS OF METHOD DEVELOPMENT AS OF 2016 34
2.2.7 DISCUSSION 35
3.1 BTBPE 39
3.1.1 Hazard Identification for BTBPE 39
3.1.2 Toxicity and Dose-Response Information on BTBPE 40
3.2 EH-TBB 40
3.2.1 Hazard Identification for EH-TBB 41
3.2.2 Toxicity and Dose-Response Information for EH-TBB 42
3.3 BEH-TEBP 43
3.3.1 Hazard Identification for BEH-TEBP 44
3.3.2 Toxicity and Dose-Response Information for BEH-TEBP 45
4 EXPOSURE ASSESSMENT 49
4.1 Available Relevant Exposure Information 49
4.2 Brominated Flame Retardants Measured in Children’s Room in Kuopio, Finland 50
5 RISK CHARACTERISATION FOR BEH-TEBP 57
6. DISCUSSION 57
7. SUMMARY AND CONCLUSIONS 61
8. REFERENCES 63
APPENDICES 67
APPENDIX 1: Reference values for BFR and OPFR analysis in house dust 67
APPENDIX 2: Criteria Used by USEPA to Assign Hazard Designation 69
6
1. INTRODUCTION
Flame retardants (FRs) are substances that can be added to polymer-based materials to
increase the materials’ resistance to ignition, and to slow down combustion. The purpose of
FRs is to reduce the risk of fire and delay the spread of fire. FRs are widely used in
commercial and consumer products due to existing fire safety regulations. Flammability tests
are required in building materials, industrial electronics and transportation vehicles
worldwide. For example, the EU Construction Products Directive stipulates flammability
standards for building materials. In the EU, the UK has set flammability standards for home
furniture. In Finland, there are flammability standards for upholstered furniture and mattresses
(EFRA 2015).
An example of required flammability standard in Finland is the EN 1021-1 & 2:2014, Decree
479/1996 for furniture upholstery. In a flammability standard test, the test fabric is exposed to
different ignition sources, specifically, a burning cigarette and butane flame, to examine its
burning behavior. The test material passes if no ignition occurs or there is only a limited area
of charring (SP 2016). As a result of flammability standards, flame retardants are ubiquitous
in the indoor environment, having been added to a wide range of industrial and commercial
products. FRs have been used in building insulation, furniture upholstery, carpet padding,
plastic casings for some electronics, and some baby products.
FRs can be emitted from these products into indoor air. The chemicals then bind to and settle
with indoor dust. FRs can accumulate in indoor dust, and it has been found that house dust
contributes to a high proportion of exposure in the indoor environment. Therefore, levels of
FRs present in house dust can be an indication of human exposure to FRs (Ni et al. 2013).
Moreover, concentration of FRs in gas phase can be estimated from measured concentrations
in house dust. For more volatile FRs, such as Pentabromotoluene (PBT), exposure through
gas phase may be significant as well (Little et al 2012).
Halogenated flame retardants and organophosphorous flame retardants make up approx. 30%
of FR used in the EU (European Union, 2011). Many of these FRs are known to be toxic,
persistent, bio-accumulative and can be transported through long distances. One example is
Polybrominated diphenyl ethers (PBDE). TetraBDE, pentaBDE, hexaBDE and heptaBDE
7
have been listed in the Annex A of the Stockholm Convention on Persistent Organic
Pollutants, whereby the production and usage of these mixtures have to be eliminated by the
parties to the Convention, for whom the amendment applies (United Nations 2015). In the
European Union, sale of commercial pentaBDE and octaBDE mixtures, in concentrations
higher than 0.1% by mass, has been banned. Under EU Directive 2002/95/EC, all new
electronic equipment must be free of PBDEs since July 2006. DecaBDE has been regulated
under Annex XVII of the European Commission Regulation EC 1907/2006 concerning the
Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), which
prohibits the use pf DecaBDE as a substance and restricts the content of DecaBDE in articles
to 0.1% (SGS 2017). All PBDEs are being regulated under the EU Restriction of Hazardous
Substances (RoHS) directive (European Union, 2011).
New BFR and OPFR mixtures have been developed to replace banned PBDEs. For example,
FM-550 and FMBZ-54 contain brominated and organophosphorous flame retardants such as
2-ethylhexyl-2,3,3,5-tetrabromobenzoate(EH-TBB) and bis-(2-ethylhexyl)-tetrabromophtalate
(BEH-TEBP). However, the toxicity of these novel and emerging FRs to human health, and
possible tendency for bio-accumulation have become a concern (Norrgran et al. 2015).
Determination of brominated and organophosphorous flame retardants in indoor dust
discussed in this thesis is based on the method developed by Van den Eede et al. 2012. The
principle aim of this method development is to develop and validate an effective analysis
method for indoor dust containing selected novel and emerging BFR, OPFR, as well as
PBDEs. Table 1 shows the BFR and OPFR that has been selected for the method
development.
8
Table 1. List of flame retardants included in method development.
CAS Number Abbreviation Full Chemical Name Molecular Weight Molecular Formula Chemical Structure
1 115-96-8 TCEP Tris(2-chloroethyl) phosphate 285.49 C6H12Cl3O4P
2 13674-84-5 TCIPP Tris(1-chloropropan-2-yl) phosphate 327.56 C9H18Cl3O4P
3 13674-87-8 TDCIPP Tris(1,3-dichloroisopropyl) phosphate 430.9 C9H15Cl6O4P
4 126-72-7 TDBPP Tris(2,3-dibromopropyl) phosphate 697.61 C9H15Br6O4P
5 126-71-6 TIBP Tri(isobutyl)phosphate 266.32 C12H27O4P
6 126-73-8 TNBP Tri-n-butylphosphate 266.32 C12H27O4P
9
7 78-51-3 TBOEP Tris(2-butoxyethyl)phosphate 398.48 C18H39O7P
8 1241-94-7 EHDPP 2-Ethylhexyl diphenyl phosphate 362.4 C20H27O4P
9 78-42-2 TEHP Tris(2-ethylhexyl)phosphate 434.64 C24H51O4P
10 115-86-6 TPHP Triphenyl phosphate 326.29 C18H15O4P
11 1330-78-5 TMPP Tris (methylphenyl) phosphate (isomer mixture) 368.37 C21H21O4P
12 3322-93-8 ab-DBE-
DBCH
alpha/beta-Tetrabromoethylcyclohexane 427.8 C8H12Br4
13 87-83-2 PBT Pentabromotoluene 486.62 C7H3Br5
10
14 35109-60-5 TBP-DBPE 2,3-Dibromopropyl 2,4,6-tribromophenyl ether 530.67 C9H7Br5O
15 183658-27-7 EH-TBB 2-Ethylhexyl-2,3,4,5-tetrabromobenzoate 549.92 C15H18Br4O2
16 37853-59-1 BTBPE 1,2-Bis(2,4,6-tribromophenoxy)ethane 687.64 C14H8Br6O2
17 26040-51-7 BEH-TEBP Bis(2-ethyl-1hexyl)tetrabromophthalate 706.14 C24H34Br4O4
11
In the second part of this thesis, a human health risk assessment for one of the above selected
BFRs, namely BEH-TEBP is performed based on currently available scientific literature and
estimated exposure from indoor dust ingestion. Risk assessment is based on the following
paradigm: hazard identification, dose-response assessment, exposure assessment and risk
characterization.
The OECD eChem portal, HSDB database and the ECHA CHEM database were consulted for
available relevant information on the above-listed BFRs, namely, ab-DBE-DBCH, PBT, TBP-
DBPE, EH-TBB, BTBPE and BEH-TEBP.
There was insufficient toxicological information available for ab-DBE-DBCH, PBT, TBP-
DBPE to perform a hazard identification. Relevant information available for the substances
BTBPE, EH-TBB and BEH-TEBP will be summarized in Section 3. The method described in
this thesis was used for the determination of BFRs from house dust samples collected around
Kuopio. An exposure estimation has been performed based on the amount of BFR detected
from a recent unpublished study by THL, where 40 house dust samples were analysed for
BFRs. Based on this exposure estimation, Risk Characterisation Ratio based on the Guidance
on Information Requirements and Chemical Safety Assessment (ECHA 2016a) has been
calculated for BEH-TEBP according to the DNEL for oral exposure.
The substance Bis(2-ethyl-1hexyl)tetrabromophthalate (BEH-TEBP) with CAS Number
26040-51-7 was therefore selected for risk characterization based on the availability of a
chemical registration dossier (ECHA 2016b) for BEH-TEBP through the European Chemical
Agency’s ECHA CHEM database, as well as a derived no-effect level (DNEL) for oral
exposure.
12
2 ANALYTICAL METHOD DEVELOPMENT FOR BFRs IN HOUSE DUST
The principle aim of this method development is to develop and validate an effective analysis
method for indoor dust containing selected novel and emerging BFR, OPFR, as well as
PBDEs. These compounds are analyzed together in the same sample. The aim of method
development is to achieve a reasonable extraction solvent volume, small sample amount
consumption, simple and easy clean-up, minimal loss of sample, technical feasibility with
available machines and techniques, short extraction time and high sample throughput.
The analytical method used throughout the method development was based on Van den Eede
et al. (2012). Gas Chromatography - Electron Impact – High Resolution Mass Spectrometry
(GC-EI-HRMS) was used to analyze the samples. The method follows the following general
protocol described in section 2.1 below.
Between May-August 2015, three development tests were done. The tests performed were a
continuation of the development tests that had been performed at THL before May 2015. The
three tests done were coded 15T024, 15T029 and 15T031.
2.1 MATERIALS AND METHODS
2.1.1 CHEMICALS AND INSTRUMENTS
Samples Used
During the course of the method development, samples used include a prepared 12C mixture,
Standard Reference Material 2585 of indoor dust, and actual samples of air filter, settled dust,
and air conditioner filter dust. In the validation test, high and low-level spiked samples were
also used as a comparison to actual samples. Typically, 50 mg of dust sample was weighed in
each test tube for extraction. One hundred μL 13C Internal Standards (2.5-6.25 ng/sample in
toluene) were added to the sample before extraction.
Florisil Clean-Up Columns
Prepared activated Florisil cleanup columns in 3 ml glass tubes were used for the clean-up
process. Florisil was activated before use in a 200°C oven overnight and cooled in a
desiccator before use. Three ml:n LiChrolut SPE tubes, of 6 cm in length, by Merck were
13
used. A small piece of cotton wool was fitted to the bottom of the tubes. Columns were
packed with 2cm layer of activated Florisil. A small cotton wool plug was added on top of
each column to prevent dusting of the Florisil when solvent was poured on top. Florisil
columns were washed with 2.5*2 ml 50% dichloromethane-hexane (dcm-hex) before
fractionation.
H2SO4-Silica Clean-up Columns
Prepared H2SO4-silica clean-up columns were subsequently added under Florisil column for
Fraction 1 only. Three ml LiChrolut SPE tubes, of 6 cm in length, by Merck were used. A
small piece of cotton wool was fitted to the bottom of the tubes. Columns were packed with 2
cm layer of 44% volume-per-weight H2SO4-silica. A small cotton wool plug was added on top
of Florisil column to prevent dusting when solvent was poured on top. H2SO4-silica columns
were washed with 2.5*2 ml 50% dcm-hex before fractionation. In the sections that follow, test
procedures are described backwards (GC-HRMS, fractionation and cleanup, extraction) as
compared to order of sample preparation (extraction, fractionation and cleanup, GC-HRMS)
because GC-HRMS method has to be in place before anything else can be tested. However,
there is naturally overlap and feedbacks between all of these phases.
2.1.2 GC-HRMS CONDITIONS
GC-MS analysis was performed with an HP 6890 gas chromatograph (Agilent, Little Falls,
DE, USA) coupled to an Autospec Ultima high resolution mass spectrometer (Waters,
Manchester, GB). The system was equipped with PTV injector and CTC CombiPal
autosampler. MassLynx 4.0 software was used for instrument control and data analysis.
Analysis of fraction 1 containing selected BFRs was as follows: empty multi baffle liner
(Agilent, Part# 5183-2037) was used in the PTV inlet. A Phenomenex ZB-5MSplus (Part No.
7FD-G030-08) capillary column cut to a length of 6 m, 0.18 mm I.D., and 0.18 µm film
thickness was used. The injection volume was 2 µl. The program for PTV splitless injection
was: 90 °C for 0.10 min, 600 °C/min to 300 °C, hold 1.0 min, 700 °C/min to 325°C, hold 7.0
min. Vent time was 0.09 min, vent flow 80 ml/min and vent pressure 100 kPa. Purge time was
1.25 min. Constant helium gas flow was 1.0 ml/min. Oven temperature program was: 70 °C
for 1.25 min, 20°C/min to 240°C, 50°C/min to 300 °C, 15 °C/min to 320 °C, hold 12.0 min.
14
For PFRs GC-HRMS analysis was as follows: a single baffle liner (Zebron, Part No: AG2-
1F06-05) with deactivated cotton wool plug in the bottom was used in the PTV inlet. An
Agilent DB-5MS UI (Part No. 122-5532UI) capillary column of 30 m, 0.25 mm I.D., and 0.25
µm film thickness was used. The injection volume was 1 µl. The program for PTV splitless
injection was: 80 °C for 0.10 min, 720 °C/min to 280 °C, hold 2.0 min, 720 °C/min to 320 °C,
hold 20.0 min. Vent time was 0.09 min, vent flow 100 ml/min and vent pressure 80 kPa.
Purge time was 2.00 min. Constant helium gas flow was 1.0 ml/min. Oven temperature
program was: 70 °C for 1.00 min, 15 °C/min to 150 °C, 10 °C/min to 280 °C, 7 °C/min to
320 °C, hold 12.0 min.
MS-parameters for both BFRs and PFRs were: MS transfer line temperature 300 °C,
temperature of ionization chamber 280 °C and energy of EI+ ionization 35 eV. The two most
intense and/or interference free ions of each compound were selected for monitoring. Sample
peak identification was based on the matching of retention times and ion ratios with those of
standards.
2.1.3 FRACTIONATION AND CLEAN-UP
Determination of Suitable Eluent
Test 15T024 was conducted on 12 May 2015. The aim of this test was to separate BFR and
OPFR each to their own fraction. This test endeavoured to determine the weakest eluent
possible to elute all OPFRs from the Florisil column. Weakest eluent implies that the elution
of impurities can be minimized. This test was performed with Florisil column only. 12-C
standards of known BFR and PFR were used for the elution test. One hundred μl of 12-C
standards, containing 10-25ng of target compounds was used in each sample. Target
compounds were added to the columns in 500μl of hexane and 1 drop of nonane. No dust
material was analyzed and therefore no extraction was performed. After fractionation, 100μl
of 13-C internal standards were added to each 12-C mixture fraction. Samples were then
concentrated and analyzed by GC-EI-MS. Table 2 shows the 12C standard mixture that was
used in this test. Table 3 shows the internal standard and recovery standard used in this test
and the subsequent tests.
15
Table 2. 12C Mixture used in Test 15T024.
16
Table 3. Internal and Recovery Standard used in Test 15T024 and subsequent tests.
17
2-Step Clean-Up
A 2-step clean-up was then performed, resulting in fractionation of analytes and removal of
interferences. Dual clean-up columns, with Florisil column on top of H2SO4-silica column
was used for Fraction 1 only. To elute Fraction 1 containing BFR (except BEH-TEBP),
extracts were added to dual-column according to the columns’ capacity, taking care not to
overflow. Samples were then eluted with 2*2.5ml of eluent 1 until all solvent had come
through both Florisil and H2SO4-silica columns. Each sample was collected in a 10ml test
tube. For Fraction 2, containing OPFR, the H2SO4-silica columns were removed, and eluate
from Florisil column was collected separately in a clean 10ml test tube. Florisil columns were
eluted with 2*2.5ml of eluent 2 until all solvent has come through.
Fraction 1 contains most of the selected BFR. Fraction 2 contains most of the selected OPFR.
The aim of the clean-up was to have most BFRs in Fraction 1 and most OPFRs in Fraction 2.
Fig. 1 Dual clean-up columns for Fraction 1, Florisil column on top of H2SO4-silica column.
Figure 1 is a photograph of the dual clean-up columns for Fraction 1, with Florisil column on
top of H2SO4-silica column.
18
Determination of Best Clean-Up Technique
Test 15T031 was conducted on 16 June 2015. This test attempted to extract selected BFRs
and OPFRs from Standard Reference Material of Indoor Dust SRM 2585. Extraction was
performed as described in Section 2.0 with 2ml of dichloromethane as the extraction solvent.
Fractionation was performed with a dual-column set-up as described above and shown in Fig.
1.
The aim of this test was to test three different fractionation techniques for the best clean-up
result and method simplicity. Each technique to be tested was added to two samples. The
following fractionation techniques were tested:
1. Direct pouring of combined 2+2ml dichloromethane extract to dual fractionation-
clean-up columns.
2. Evaporation of combined 2+2ml dichloromethane extract to 1ml dichloromethane
before adding to dual-columns.
3. Evaporation of combined 2+2ml dichloromethane extract to 0.5ml dichloromethane,
and dilution with 0.5ml hexane before adding to dual-columns.
Dual clean-up columns, with Florisil column on top of H2SO4-silica column were used for
Fraction 1 only. For Fraction 1, containing BFRs, extracts were added to each dual-column
according to the columns’ capacity, taking care not to overflow. Samples added with above-
listed techniques 1 and 2 were eluted with 2*2.5ml of dichloromethane until all solvent has
come through both Florisil and H2SO4-silica columns. Samples added with above-listed
technique 3 were eluted with 2*2.5ml 50% dichloromethane-hexane. Each sample was
collected in a 10ml test tube.
For Fraction 2, containing OPFR, the H2SO4-silica columns were removed, and elute from
each column was collected separately in a clean 10ml test tube. Florisil columns were eluted
with 2*2.5ml of 10% acetone-dichloromethane until all solvent has come through.
After fractionation and clean-up, 30μL of nonane was added to each sample. Elutes were then
concentrated and analyzed as described in this section.
19
Na2SO4 clean-up of Fraction 2
In Test 15T029, which will be elaborated in Section 2.1.4, interferences in the MS
chromatograms were observed in Fraction 2. Eluates for Fraction 2 for Test 15T029, which
had also been concentrated and transferred to autosampler vials, went through a further clean-
up step with Na2SO4. One ml of hexane was first added to each autosampler vial containing
FR compounds in 120μL of toluene and 30μL of nonane. The contents of each vial were then
transferred to a 5ml test tube with a Pasteur pipette. Each autosampler vial was then flushed
with a further 1.0ml of hexane, which was also transferred to the corresponding test tube.
Approximately 500μg of activated Na2SO4 was added to each test tube. Test tubes were then
shaken in the VWR-shaker for 4 minutes at 1800rpm in pulse mode (3 seconds on, 1 second
off). At this point, the yellow colour was reduced but not completely removed. Samples were
centrifuged in 3000rpm for 2*2 minutes. The hexane (containing FR) in each test tube was
then decanted to a clean 5ml test tube. Shaking with VWR and centrifuging were then
repeated on the sample containing Na2SO4. The two portions of hexane from each sample
were then combined in the same clean test tube.
Concentration and GC-MS Analysis
After fractionation and clean-up, 30μL of nonane was added to each sample. Elutes were
concentrated under a gentle stream of nitrogen gas to a volume of about 500μL before transfer
to GC-MS autosampler vials. Each test tube was flushed with 2*200μL of dichloromethane to
ensure complete transfer of target compounds to autosampler vial. Fifty μL of 13C Recovery
Standards (5 – 10 ng/sample in hexane) were added before GC-MS analysis. Thirty μL of
nonane was also added to each autosampler vial. (No additional nonane would be added if
nonane had already been added to samples before evaporation.) Vials were covered with
wadding and evaporated in fume hood overnight. Cleaned and concentrated extracts were then
analyzed in mixture of 120μL of toluene and 30μL of nonane.
20
2.1.4 EXTRACTION
Extraction of FR from Dust Sample
Extraction was done by putting the sample at 1800 rpm to VWR Shaker for 4 minutes in a
pulse mode of 3 seconds on, 1 second off, followed by 3500 rpm centrifugation for 2 min.
Two ml of extraction solvent was used for each dust sample. Extraction was then repeated
with a further 2ml of extraction solvent. 2+2 ml of extract for each sample were then
combined.
During the method development, different extraction and elution solvents were tested, namely
hexane, dichloromethane, acetone, diethylether and their appropriate mixtures.
Determination of Suitable Extraction Solvent
Test 15T029 was conducted on 26 May 2015. This test attempted to extract selected BFRs
and OPFRs from the intended medium. Standard Reference Material of Indoor Dust SRM
2585 was used as samples for this test. The aim of this test was to determine a suitable
extraction solvent. Three different solvents: 100% Dichloromethane, 25% Acetone-Hexane,
50% Dichloromethane - Ether were tested in this test. Both 100% Dichloromethane and 25%
Acetone-Hexane were determined to be suitable extraction solvents for polar BFRs and
OPFRs by Van den Eede et al. (2012). 50% Dichloromethane-Ether was a new solvent to be
tested. It was noted that the ether had to be carefully evaporated away for the H2SO4-silica
clean-up applied to the OPFR fraction.
2.1.5 VALIDATION
A validation was subsequently performed for the method development. This validation
evaluates the performance of the analysis of brominated compounds. Performance of the
analysis of organophosphorous compounds is also evaluated, and possible further
developmental needs identified, especially in sample clean-up.
Table 4. shows the type and description of the samples used for the validation test. Samples
were prepared and analysed according to the method previously described.
21
Table 4. Type and description of the samples used for the validation test.
Blank No sample. Only extraction solvent 100%
Dichloromethane
National Institute of Standards and Technology (NIST)
SRM-2585
Standard Reference Material – 2585 House
Dust
Home exhaust air filter dust from a private home (Home 1) Indoor dust collected from exhaust air filter
with a vacuum cleaner. Dust accumulated
through several months. Collected on 6
January 2015 and 24 July 2015 and mixed.
Home settled dust from a private home (Home 1) Indoor dust collected from home surfaces
with a vacuum cleaner. Collected on 25 July
2015.
Home air condition filter dust
rom a private home (Home 2)
Indoor dust collected from air condition
filter dust with vacuum cleaner. Collected
on 29 July 2015.
Low-level spiked home exhaust air filter dust Home exhaust air filter dust spiked
High-level spiked home exhaust air filter dust Home exhaust air filter dust spiked
Preparation of Spiking Solution and Spiking of House Dust
Spiked house dust is prepared from dust collected from home exhaust air filter dust of a
private home (described above).
100% dichloromethane was used as spiking solvent. Approximately 6ml of spiking solvent
and an appropriate volume of BFR stock solution was added to 1.0g of dust in an Erlenmayer
flask and mixed in VWR-shaker at 500rpm for 30 minutes. The flask was uncapped and
solvent let evaporate overnight at room temperature in fume hood, until the original weight of
the dust sample had been achieved. The spiked dust was then re-homogenized by shaking in
VWR-shaker for 3 minutes at 2500 rpm. One g of low level spiked sample, and another 1.0g
of high level spiked sample were prepared. Tables 5 and 6 below show the concentration of
each compound in the stock solution and the final spiked concentration to the dust.
22
Table 5. FR concentration in the stock solution and concentration of FR spiked to the low
level spiked sample.
Compound
Concentration in
the stock solution
(ng/ml)
Pipet to test
tube containing
dcm (µl)
Mass in
test tube
(ng)
Concentration
spiked to the
dust (ng/g)
TCEP 100
100
10 10
TCIPP 100 10 10
TDCIPP 100 10 10
TDBPP 100 10 10
TiBP 100 10 10
TnBP 100 10 10
TBOEP 250 25 25
EHDPP 100 10 10
TEHP 100 10 10
TPHP 100 10 10
TMPP 100 10 10
ab-DBE-
DBCH 200 20 20
PBT 100 10 10
TBP-DBPE 100 10 10
EH-TBB 250 25 25
BTBPE 100 10 10
BEH-TEBP 100 10 10
DBDPE 250 25 25
BDE-28 50
100
10 10
BDE-47 50 10 10
BDE-99 50 10 10
BDE-100 50 10 10
BDE-153 35 7 7
BDE-154 50 10 10
BDE-183 50 10 10
BDE-209 160 250 40 40
23
Table 6. FR concentration in the stock solution and final concentration of FR spiked to the
high level spiked sample.
Compound
Concentration in
the stock solution
(ng/ml)
Pipet to test
tube containing
dcm (µl)
Mass in
test tube
(ng)
Concentration
spiked to the
dust (ng/g)
TCEP 100
1000
100 100
TCIPP 100 100 100
TDCIPP 100 100 100
TDBPP 100 100 100
TiBP 100 100 100
TnBP 100 100 100
TBOEP 250 250 250
EHDPP 100 100 100
TEHP 100 100 100
TPHP 100 100 100
TMPP 100 100 100
ab-DBE-DBCH 200 200 200
PBT 100 100 100
TBP-DBPE 100 100 100
EH-TBB 250 250 250
BTBPE 100 100 100
BEH-TEBP 100 100 100
DBDPE 250 250 250
BDE-28 50
1000
100 100
BDE-47 50 100 100
BDE-99 50 100 100
BDE-100 50 100 100
BDE-153 35 70 70
BDE-154 50 100 100
BDE-183 50 100 100
BDE-209 160 2500 400 400
24
In the validation, the following parameters were determined:
• Limit of Detection (LoD) = 3*SD of blank sample
• Limit of Quantification (LoQ) = 8*SD of blank sample
• Accuracy (as compared with reference values and other labs)
• Precision (relative standard deviation for each compound)
• Measurement Uncertainty (based on accuracy and precision)
• Suitability of chromatographic system
Determination of Limit of Detection (LOD) and Limit of Quantification (LOQ)
Limit of detection and limit of quantification were calculated as follows:
LOD = 3 * SD
LOQ = 8 * SD
SD is the standard deviation of the blank samples producing detectable signal with correct ion
ratios, i.e. +/- 20% of which in calibration samples or theoretical values. If no signal from
blank sample is obtained, LOD and LOQ were calculated as 95th percentile of the
concentrations corresponding to signal to noise ratios – 3:1 for LOD and 8:1 for LOQ,
produced by MassLynx software from all actual samples.
Accuracy and Precision
Accuracy was determined as the percent deviation of the analysis results SRM-2585 from the
certified values. Comparison was more qualitative for compounds with only reference values
from other laboratories. Percent recovery from spiked samples was also used to estimate
accuracy. Precision was calculated as the relative standard deviation of the results from all
dust samples.
Accuracy -1: For 2 batches of actual samples, 4 parallel samples of SRM-2585 were measured
and their results compared to certified or referenced values.
Accuracy – 2: For 2 batches of samples, 4 parallel samples of spiked dust were measured and
percentage recovery was calculated for all compounds as follows:
25
% Recovery = 100*(Average results of spiked samples – Average of non-spiked results)/
Spiked concentration
Precision: Relative standard deviation was calculated separately from parallel samples for
each different house dust sample. Combined precision was calculated for each compound as
the root mean square of Relative Standard Deviation - % from all different dust samples. If
large deviations in the range of concentrations occur, precision can be calculated for different
ranges of concentrations.
Measurement Uncertainty
Measurement uncertainty was calculated as a combination of accuracy and precision.
Measurement Uncertainty: (utot)2=(uAccu)2+(uPres)2
Expanded Measurement Uncertainty with 95 % confidence interval
MU=2*utot
utot = Total MU
uAccu = Accuracy
uPres = Precision
Suitability of the Chromatographic System
This validation aimed to have chromatographic peaks that are symmetrical and without
interfering extra peaks in the channel of the compound to be measured. Also, there was to be
no serious interferences in the HRMS lock masses at the retention times of compounds to be
measured. Recovery rates of internal standards would preferably be in the range between 60%
and 120%. Lower recovery rates would be acceptable for highly volatile compounds. Ion
ratios of compounds to be measured should be within +/- 20% of the theoretical values or
values obtained from calibration standards.
26
2.2 RESULTS AND DISCUSSION
2.2.1 FRACTIONATION AND CLEAN-UP OPTIMIZATION
Results for Test 15T024
In this test, the standard 12C mixture of compounds in 500 μl of hexane were added to cleanup
columns. It was concluded that 100% Dcm was the most suitable solvent for the elution of
Fraction 1 from the Florisil column. With this solvent most of the BFRs could be separated
from the OPFRs. Ten% Acetone-Dcm was determined to be the most suitable solvent for the
elution of Fraction 2, where all of OPFR, could be eluted, except that 50% of TBOEP still
remained in the column. However, the use of 13C-labelled internal standard for TBOEP
corrects for this loss. BEH-TEBP was eluted in Fraction 2 together with OPFR, and would
remain so in subsequent tests.
Results for Test 15T031
In this test it was found that the detected concentration of each compound was quite consistent
across all 3 treatments. Table 7 below illustrates the results for this test.
Table 7. The detected concentration of each compound was quite consistent across all 3
treatments. BFR Extract (4 ml of
DCM) directly
to dual-column
Extract evaporated
to 1mL then to
dual-column
Extract evaporated to
0.5ML, 0.5mL hex added
then to dual-column
Certified/reference
values (range)
ng/g
Average Average 15T029-6-F1
ng/g ng/g ng/g
ab-DBE-DBCH 6 3 3
PBT 0 0 0
TBP-DBPE 0 0 0
EH-TBB 17 20 21 26-40 (2-6)
BTBPE 15 14 20 32-76 (4-14)
BEH-TEBP 1898* 6216* 0* 145-1300(16.7-
94)*
DBDPE 0 0 0 <20
BDE-28 24 24 25 46.9 (4.4)
BDE-47 243 249 279 497 (46)
BDE-100 74 78 95 145 (11)
BDE-99 486 502 577 892 (53)
BDE-154 40 41 49 83.5 (2.0)
BDE-153 59 60 68 119 (1)
BDE-183 no data no data no data 43.0 (3.5)
BDE-209 2306 2241 2336 2510 (190)
* It was found that BEH-TEBP had degraded in H2SO4-silica , therefore, these results for BEH-TEBP for Fraction 1 were not reliable.
27
The average concentration of compounds for 15T031 detected was approximately 50% of
certified/reference values. This error was due to calibration and was corrected in subsequent
tests. However, in the interest of determining the most simple and suitable fractionation
technique, the above technical issues were not pursued further.
In Test 15T029, activated Na2SO4 was found to be ineffective in removing impurities in
Fraction 2. Tests for the cleanup of Fraction 2 were not continued further.
2.2.2 EXTRACTION
Results for Test 15T029
The above-described clean-up facilitated the GC-HRMS analysis of Fractions 1-A and 1-B.
Based on the finding that the detected concentration of BFR and PBDE were consistent across
all three treatments in Test 15T031, direct pouring of 4ml Dcm extract was chosen for future
implementation. As Table 8 shows, the concentrations for PBDEs analyzed from SRM2585
by different solvents was generally consistent with certified values.
Table 8. The concentrations for PBDEs analyzed from SRM2585 by different extraction
solvents. Certified Value (SRM-
2585)
100%
Dichloromethane
25% Acetone-
Hexane
50%
Dichloromethane
-Ether
ng/g Average (ng/g) Average (ng/g) Average (ng/g)
BDE-28 46.9 (4.4) 45 46 45
BDE-47 497 (46) 531 519 524
BDE-100 145 (11) 175 170 173
BDE-99 892 (53) 1019 1009 1034
BDE-154 83.5 (2.0) 83 81 83
BDE-153 119 (1) 114 111 112
BDE-183 43.0 (3.5) 42 42 36
BDE-209 2510 (190) 2570 2871 2549
All three of the elution solvents tested, namely, 100% Dichloromethane, 25% Acetone-
Hexane, 50% Dichloromethane - Ether were shown to be equally efficient in extraction of
BFR. Table 9 shows that the amount of BFRs extracted are comparable across all three
elution solvents.
28
Table 9. The amount of BFRs extracted are comparable across all three elution solvents.
BFR Comparison value
(range)
100%
Dichloromethane
25%
Acetone-
Hexane
50%
Dichlorometh
ane-Ether
Average (ng/g) Average
(ng/g)
Average (ng/g)
ab-DBE-
DBCH
5 3 3
PBT 0 0 0
TBP-DBPE 0 0 0
EH-TBB 26-40 (2-6) 38 38 39
BTBPE 32-76 (4-14) 50 33 33
BEH-TEBP 145-1300(16.7-94) n/a n/a n/a
DBDPE <20 0 1 1
100% dichloromethane was chosen to be the most suitable extraction solvent for subsequent
extractions due to ease of preparation and use. It was found that an additional H2SO4- silica
clean-up column was necessary for the BFR fraction in order for the extract to be sufficiently
clean for the GC-HRMS analysis. The main benefit of using 100% dichloromethane is that it
can be directly poured to dual Florisil - H2SO4- silica clean-up column after extraction
without prior evaporation that is needed for 25% Acetone-Hexane and 50% Dichloromethane-
Ether.
29
2.2.3 SUMMARY OF SAMPLE TREATMENT IN FLOW CHART
Fig. 2 Flow-chart of analysis method.
Figure 2 shows a flow-chart of the analysis method.
2.2.4 RESULTS OF VALIDATION
Summary of Results
LOQ & MU were acceptable for all BFRs. LOQ of BFRs ranged between 0.5 – 5.0 ng/g. For
OPFR, LOQ ranged between 6.9-613 ng/g. Precision for OPFR was good, meaning the
relative SD were consistent for each compound. Accuracy for OPFR was poor compared to
other labs’ analysis result for the certified material for indoor dust SRM 2585. Therefore,
method for OPFR analysis requires further testing that was not conducted within the scope of
this thesis. Tables 10, 11, 12 and 13 below present a summary of validation results, taken
from an internal validation report written by Panu Rantakokko, PhD, Senior Researcher of
THL on 11 September 2015.
30
Table 10. Summary of LODs, LOQs and MUs to be reported for BFRs.
Compound LOD (ng/g) LOQ (ng/g)
MU (%)
< 50 ng/g
MU (%)
> 50 ng/g
ab-DBE-DBCH 1.0 2.5 55 55
PBT 0.2 0.5 55 40
TBP-DBPE 0.2 0.5 65 45
EH-TBB 2.0 5.0 25 25
BTBPE 0.2 0.5 25 25
BEH-TEBP* 10 25 100 50
DBDPE 2.0 5.0 30 30
BDE-28 0.2 0.5 25 20
BDE-47 0.2 0.5 30 20
BDE-100 1.0 2.5 40 40
BDE-99 1.0 2.5 25 25
BDE-154 0.3 0.7 25 25
BDE-153 0.3 0.7 30 30
BDE-183 0.6 1.5 35 35
BDE-209 1.0 2.5 75 75
Table 11. LODs and LOQs based on blank (n=4) and MassLynx (OPFRs, n=21).
Compound
/Sample LOD (ng/g) LOQ (ng/g)
TIBP 20 52
TNBP 12 32
TCEP 25 67
TCIPP 230 613
TDCIPP 21 57
TPHP 2.6 6.9
TBOEP 6.5 17
EHDPP 34 91
TEHP* 40 106
TMPP 7.1 19
*TEHP has very poor sensitivity with the ions selected, but alternative ions would be low
mass and extremely noisy and non-specific.
31
Table 12. Accuracy, precision and MU from results of SRM 2585 a (BFRs, n=8).
COMPOUND
Average (range)
(ng/g)
RSD
(%)
Certified/Other
(ng/g)
Recovery
(%) MU (%) Source
ab-DBE-DBCH <LOQ
PBT 0.22 (0.12-0.26) 24
TBP-DBPE <LOQ
EH-TBB 33 (31-36) 5.0 26 127 56 Van den Eede 2012
BTBPE 54 (35-86) 40 b 39 140 b 112 b Van den Eede 2012
BEH-TEBP c 1212 (1141-1281) 5.6 574 211 c 223 c Van den Eede 2012
DBDPE <LOQ <7.1 Van den Eede 2012
BDE-28 49 (47-51) 3.5 46.9 104 11 Certified
BDE-47 487 (443-551) 8.2 497 98 17 Certified
BDE-100 164 (148-191) 9.5 145 113 32 Certified
BDE-99 954 (870-1114) 8.5 892 107 22 Certified
BDE-154 83 (78-96) 7.6 84 99 15 Certified
BDE-153 115 (109-133) 7.5 119 96 17 Certified
BDE-183 37 (33-46) 11 43 87 34 Certified
BDE-209 3138 (2368-4807) 26 d 2510 125 d 72 d Certified a SRM 2585 has certified concentrations for PBDEs only. For other BFRs many sources exist, but Van den
Eede represents generally a recognized high quality laboratory. b For BTBPE some results were outliers, especially in the series 2. Source of deviation needs to be tested in
future work. c Sahlström et al 2012 measured a concentration of 1300 ng/g for BEH-TEBP. d Range of results for BDE-209 is large. Possible laboratory contamination needs to be tackled in future
testing.
Table 13. Accuracy, precision and MU from results of SRM 2585 a (OPFRs, n=4).
COMPOUND
Average
(range) (ng/g)
RSD
(%) b
Certified/
Other (ng/g)
Recovery
(%) b
MU
(%) Source
TIBP 18 101.5
TNBP 1196 1.6 190 629 1059 Van den Eede 2012
TCEP 3363 6.2 680 495 789 Van den Eede 2012
TCIPP 3780 9.8 860 440 679 Van den Eede 2012
TDCIPP 7744 14.0 3180 244 288 Van den Eede 2012
TPHP 4335 5.9 1160 374 548 Van den Eede 2012
TBOEP 57568 7.5 63000 91 23 Van den Eede 2012
EHDPP 3039 9.5 1300 234 268 Bergh 2012
TEHP 1091 9.1 370 295 390 Bergh 2012
TMPP 3435 10.0 1140 301 403 Van den Eede 2012
32
Suitability of Chromatographic System
For the BFRs, percentage recovery of internal standards were found to be in the range of 60%
- 120%. Ion ratios of compounds to be measured were within +/- 20% of the theoretical
values, or values from calibration standards.
For the OPFRs, peak sizes tended to be too small in the calibration standard for all
compounds, and for some compounds in the Internal Standard solution added to samples.
Table 14 below illustrates the amount of dust detected in Home Settled Dust samples (n=4),
Home Exhaust Air Filter Dust samples (n=4) and Home Air Condition Filter Dust samples
(n=4) during the validation process. Home settle dust samples and home exhaust air filter dust
samples are from the same home (Home 1), while Home air condition filter dust samples are
from another home (Home 2).
33
Table 14. Amount of dust detected in Home Settled Dust samples, Home Exhaust Air Filter
Dust samples, and Home Air Condition Filter Dust samples during validation. Vapor pressure
of each compound is included for comparison. Sample
type
Settled Dust (n=4,
Home 1)
Exhaust Air Filter Dust
(n=4, Home 1)
Air Condition Filter Dust
(n=4, Home 2)
Compound Average
(ng/g)
RSD
(%)
Average (ng/g) RSD
(%)
Average (ng/g) RSD
(%)
Vapour
Pressure
(Pa)(25C)
ab-DBE-
DBCH
0.56 29.2 0.41 22 0.31 5.8 2.97E-03
PBT 0.70 11 0.63 36a 1 19 6.00E-04
TBP-
DBPE
0.64 4.9 2.0 5.4 0.08 13 1.26E-05
EH-TBB 5.6 15 8.7 3.4 2.7 23 3.71E-07
BTBPE 3.5 6.1 2.7 9.2 8.5 135 3.88E-10
BEH-
TEBP
136 19 138 8.2 192 11 1.55E-11
DBDPE 158 17 212 5.7 327 140 n/a
BDE-28 0.95 5.1 1.2 6 0.27 8.1 n/a
BDE-47 23 4.3 24 2.7 4.1 4.1 n/a
BDE-100 4.6 3.9 4.5 10.7 0.94 7.7 n/a
BDE-99 41 2.9 38 2 5.3 11 n/a
BDE-154 2.4 5.6 2.7 1.9 0.49 31 n/a
BDE-153 5.6 7.2 8.0 3.9 1.2 60 n/a
BDE-183 1.3 14 2.2 37 2.9 98 n/a
BDE-209 752 2.2 785 3.6 184 36 n/a
34
2.2.5 CONCLUSIONS OF METHOD DEVELOPMENT AS OF 2016
It was concluded that 100% Dcm was the most suitable solvent for the elution of Fraction 1
from the Florisil column. With this solvent most of the BFRs could be separated from the
OPFRs. 10% Acetone-Dcm was determined to be the most suitable solvent for the elution of
Fraction 2, where all of OPFR, could be eluted, except that 50% of TBOEP still remained in
the column. BEH-TEBP was eluted in Fraction 2 together with OPFR. Direct pouring of 4ml
Dcm extract was chosen for future implementation. 100% dichloromethane was chosen to be
the most suitable extraction solvent for subsequent extractions due to ease of preparation and
use. Settled dust was considered to be a preferable matrix.
For the BFRs, percentage recovery of internal standards were found to be in the range of 60%
- 120%. Ion ratios of compounds to be measured were within +/- 20% of the theoretical
values, or values from calibration standards. For the OPFRs, chromatography peak sizes
tended to be too small in the calibration standard for all compounds, and for some compounds
in the Internal Standard solution added to samples.
For the validation, Limit of Quantification (LOQ) & Measurement Uncertainties were
acceptable for all BFRs. LOQ of BFRs ranged between 0.5 – 5.0 ng/g. For OPFR, LOQ
ranged between 6.9-613 ng/g. Precision for OPFR was good, but accuracy was poor compared
to other labs’ analysis result for the certified material for indoor dust SRM 2585. Therefore,
method for OPFR analysis requires further testing that was not conducted within the scope of
this thesis.
As compared to sampling of settled dust by collecting settled dust on surfaces with a vacuum
cleaner, indoor dust collected on the exhaust air filter could be a good time and space
integrated sample from the entire indoor space of a household, in the case where this
particular exhaust mechanism is installed. Therefore, exhaust filter dust can be representative
of one indoor compartment as all air that exit the house goes through the filter. However, the
concentration of more volatile BFRs such as ab-DBE-DBCH and PBT tend to be lower than
that on settled dust, as illustrated in Table 14. Therefore, settled dust would be a preferable
matrix than exhaust dust within the scope of this particular validation test described in this
thesis. Air conditioner filter dust showed large deviation, as illustrated by the high relative
standard deviation in Table 14. Therefore, air conditioner filter dust was not an ideal matrix.
35
Figure 3 shows a schematic of an example of a heat recovery unit of a house ventilation
system with Stale air from inside filter used for sample collection.
Figure 3. Schematic illustration of exhaust ventilation. This figure has been provided by Dr.
Panu Rantakokko of the National Institute for Health and Welfare (THL), Kuopio.
It was noticed that the spiked samples were more homogenous than non-spiked samples when
1g of sample was spiked at one time. The reason may be that the FRs in the samples have
been extracted and equally redistributed during the spiking process with dichloromethane,
resulting in a more homogeneous overall distribution of the FRs present in the sample.
However, it would be difficult to obtain such a large quantity of indoor dust sample from a
single site unless taken from exhaust air filter. Large dust mass available from the filter is one
significant benefit of using it for dust sampling.
2.2.7 DISCUSSION
It was recommended that elution of BEH-TEBP need to be tested further to reduce BEH-
TEBP degradation in H2SO4-silica clean-up column. There was an aim to get BEH-TEBP to
36
Fraction 1. However, it has since proven to be unsuccessful. OPFR accuracy needs to be
improved, but at the present, difficulties with impurities remain. There may be a need to
increase OPFR concentrations in Internal Standard and Calibration Standard Solutions to
match the levels found in actual samples. Also, it may be good to have separate calibration
solutions for BFR and OPFR.
3. HUMAN HEALTH RISK ASSESSMENT
According to the definition put forward by the United States Environmental Protection
Agency (USEPA 2017), human health risk assessment is a process of estimating the
probability of human health effects, especially adverse health effects for a given exposure to a
substance, which can be a chemical present in an environmental medium. There are four basic
steps to human health risk assessment: hazard identification, dose-response assessment,
exposure assessment and risk characterization. Hazard identification attempts to determine
whether a substance may cause harm to humans by putting together available information on
toxicokinetics and possible adverse effects of a chemical on human health. Dose-response
assessment attempts to determine a numerical relationship between exposure to the substance
and the effects. The dose-response relationship links the probability and severity of adverse
health effects to the level of exposure to the substance. Exposure assessment is a process of
determining the magnitude of exposure, frequency of exposure and duration of exposure to
the substance. Exposure assessment also takes into consideration the population exposed to
the substance. Risk characterization summarises the information gathered for the first three
steps of the risk assessment process, and from these information, conclusions may be drawn
regarding the extent of risk resulting from exposure to a substance.
Figure 4 below shows a schematic representation of the four steps to human health risk
assessment. This figure was taken from the USEPA website (USEPA 2017).
37
Figure 4. The four-step process to human health risk assessment.
Hazard characterization in this section will be based on currently available information from
literature. The endpoint Derived No-Effect Level (DNEL) will be employed. Risk
Characterisation Ratio will be used to perform hazard characterization.
The DNEL is a part of human health hazard assessment stipulated in Annex I of REACH,
which comprises of four steps: evaluation of non-human information, evaluation of human
information, classification and labelling, and finally, the derivation of DNEL (Munn 2007).
DNEL is the level of human exposure that should not be exceeded. In hazard characterization,
the estimated exposure of a population is compared with the corresponding DNEL. The risk
of adverse health effect is considered to be adequately controlled if the exposure level does
not exceed the corresponding DNEL, as stipulated in REACH Annex I, Section 6.4 (ECHA
2016a).
The Risk Characterisation Ratio (RCR) is a way to quantify risk in a given exposure scenario.
Based on the guidance from ECHA (2016a), the RCR is calculated as follows:
RCR = Exposure/DNEL
If exposure is less than DNEL, i.e. RCR<1, the risk is adequately controlled. If exposure is
larger than DNEL, i.e. RCR>1, the risk is not adequately controlled.
38
This section will focus on the six novel and emerging BFR listed in Table 1, namely, ab-
DBE-DBCH, PBT, TBP-DBPE, EH-TBB, BTBPE and BEH-TEBP. The OECD eChem
portal, HSDB database and the ECHA CHEM database were consulted for available relevant
information on the above substances.
There was insufficient toxicological information available for ab-DBE-DBCH, PBT, TBP-
DBPE to perform a hazard identification. There is some information available for BTBPE
from the Scientific Opinion on Emerging and Novel Brominated Flame Retardants (BFRs) in
Food, issued by the European Food Safety Authority in 2012. An evaluation of human-health
related toxicity has been included in the United States Environmental Protection Agency
Alternatives Assessment update (2015) for EH-TBB and BEH-TEBP. However, the substance
EH-TBB is not found on the OECD eChem portal. Chemical registration dossier (ECHA
2016b) is available for BEH-TEBP through the European Chemical Agency’s ECHA CHEM
database. In this dossier, a Derived no-effect level (DNEL) for oral exposure has been
estimated for BEH-TEBP.
This section summarises relevant toxicological information available for the substances
BTBPE, EH-TBB and BEH-TEBP. An exposure estimation has been performed based on the
amount of BFR detected from a recent unpublished study by THL. Based on this exposure
estimation, Risk Characterisation Ratio based on the Guidance on Information Requirements
and Chemical Safety Assessment (ECHA 2016a) has been calculated for BEH-TEBP
according to the DNEL for oral exposure.
The substance Bis(2-ethyl-1hexyl)tetrabromophthalate (BEH-TEBP) with CAS Number
26040-51-7 was selected for hazard characterization based on the availability of a DNEL
value for oral exposure for the general population.
The DNEL value for BEH-TEBP has been derived for long-term oral exposure according to a
repeated dose oral toxicity study which will be described in Section 3.3.2.
A commercial mixture FM-550 containing EH-TBB and BEH-TEBP has been used in several
toxicological experiments mentioned in this section. The exact composition of FM-550 is
39
proprietary. However, it has been found by Stapleton et al. (2008) that FM-550 contains
approximately 50% of isopropylated triaryl phosphate and triphenylphosphate. The other 50%
consisted of brominated compounds EH-TBB and BEH-TEBP in approximately 4:1 ratio by
mass.
Another commercial mixture, FMBZ-54 has also been used in several experiments mentioned
in this section. FMBZ-54 comprises of EH-TBB:BEH-TEBP in an approximately 4:1 ratio
(Bearr et al. 2012).
3.1 BTBPE
The European Food Safety Authority has published a Scientific Opinion on Emerging and
Novel Brominated Flame Retardants (BFRs) in Food, updated on 6 December 2013. It was
concluded that 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), with the CAS Number
37853-59-1, has a possibility to raise concerns for bioaccumulation, based on available
experimental and environmental behavior data. BTBPE was considered to be of high
persistency in the environment. In the European Union, BTBPE has been classified as low
production volume (LPV) chemical, which implies that the import or production volume is
more than 10 tonnes but less than 1000 tonnes per year. BTBPE is in pre-registration under
REACH. However, no registration dossier has been made publicly available. There is very
limited information on toxicity in humans. This section will focus on relevant information on
possible toxicity in humans, therefore, ecotoxicological information will not be discussed.
3.1.1 Hazard Identification for BTBPE
Toxicokinetic information
In a study of rats given 0.05-5% 14C-BTBPE in the diet for one day by Nomeir et al (1993),
there was a very limited amount of radioactivity eliminated in urine, of less than 1% of dose
ingested, but a high percentage of faecal excretion, of 80-100% of the dose ingested. In most
of the tissues, there were undetectable levels of radiolabeled compounds. This suggested that
BTBPE gastrointestinal absorption is poor in rats. However, in rats given a diet with 500
mg/kg bw/day 14C-BTBPE for a duration of 10 days, it was found that the adipose tissue,
kidney, skin, the thymus contained the highest concentration. Less than 0.01 % of the dose
was found in the majority of the tissues.
40
In another study by Hakk et al. (2004), rats were given a single dose of 2 mg/kg bw 14C-
BTBPE by gavage. 100% of the dose was recovered in the faeces. The same research group
also demonstrated that elimination of radioactivity by bile was less than 1%, which suggested
that the faecal elimination was primarily from unabsorbed BTBPE. Due to this low level of
absorption, tissue level of BTBPE was low. After 72 h of the single dose given, more than
0.1% of the dose was found only in the gastrointestinal tract and carcass.
3.1.2 Toxicity and Dose-Response Information on BTBPE
LD50 for BTBPE was estimated to be >10g/kg bw for rats and dogs. No compound-related
effects were observed in rats after being fed up to 10% BTBPE in the diet, at an estimated
concentration of 35 mg/kg bw/day, for 14 days. In an inhalation study, rats inhaled BTBPE at
5 or 20 mg/liter in the atmosphere for 21 days. No gross pathological changes were observed.
However, it was observed in the lungs unspecified histopathological lesions (Matthews 1984,
cited by Nomeir et al. 1993).
The reproductive and developmental toxicity of BTBPE was studied by Egloff et al. (2011). It
has been found that there are no hatching effects in chicken. In the WHO/IPCS evaluation
(2005) for genotoxicity and carcinogenicity, BTBPE was found to be not mutagenic in Ames
test and S. cerevisiae. No information was available for BTBPE on human health endpoints.
3.2 EH-TBB
In a report published in August 2015, The USEPA has evaluated a number of novel and
emerging brominated flame retardants that have been used as alternatives for phased-out
PBDEs. 2-Ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) with CAS Number 183658-27-7
was one of the brominated flame retardants evaluated. EH-TBB is not found on the OECD
eChem portal. EH-TBB is not registered under REACH, and no information on production
volume in the EU is available (EFSA 2013).
USEPA has stipulated a hazard criteria used to interpret available data and assign a hazard
level. These hazard criteria, named the “Design for the Environment Alternatives Assessment
41
Criteria for Hazard Evaluation” by USEPA were finalized in 2011. When insufficient
information is available, hazard designation would be assigned conservatively by weight of
evidence (USEPA 2015). The criteria used by USEPA to assign hazard designations is
included as a table in Appendix 2.
3.2.1 Hazard Identification for EH-TBB
Toxicokinetic Information
Experimental data with FM-550 showed that it was possible for EH-TBB to be absorbed after
oral exposure from gestation and through lactation. EH-TBB was found in the tissues of
exposed dams and pups after exposure to the FM-550 (Patisaul et al. 2013). In a study by
Patusaul et al. (2013), pregnant rats were given 0, 0.1 or 1 mg/kg bw/day FM-550 in the diet
through gestation day 8 until post-natal day 21. FM550 components, including EH-TBB and
BEH-TEBP were detected in adipose, liver, and muscle tissues of dams at post-natal day 21 at
768 ng/g w.w. for high dose, and 29.6 ng/g w.w for low dose, and less than 7 ng/g w.w. in
controls. EH-TBB was also detected in pooled post-natal day 21 pup adipose tissue.
The primary metabolite of EH-TBB was found to be tetrabromobenzoic acid (CAS number
27581-13-1) by in vitro metabolism experiments with human liver microsomes, rat liver
microsomes, rat cytosol, rat intestinal microsomes, and rat serum following exposure to EH-
TBB. Phase two metabolites of tetrabromobenzoic acid was not found (Roberts et al. 2012).
Tetrabromobenzoic acid was also detected in liver tissue of dams on post-natal day 21 in the
experiment by Patusaul et al. (2013).
Hazard Identification
EH-TBB was evaluated by USEPA, according to the hazard criteria mentioned above and
included in Appendix 2, to have low acute toxicity and low genotoxicity. However, it was
evaluated to have a moderate carcinogenicity, moderate reproductive toxicity, developmental
toxicity, neurotoxicity and repeated dose toxicity. EH-TBB was considered to have a high
persistence and high tendency for bioaccumulation in the environment.
42
3.2.2 Toxicity and Dose-Response Information for EH-TBB
For Acute Mammalian Toxicity, the Acute Oral Lethality was estimated to be LD50 >5000
mg/kg bw based on several studies. EH-TBB was estimated to have uncertain potential for
carcinogenicity, based on professional judgement and analogy with closely related chemicals.
EH-TBB is considered to have a low genotoxicity by USEPA. Gene Mutation test in vitro and
Chromosomal Aberration in vitro tests yielded negative results (Chemtura 2006).
Reproductive Toxicity
EH-TBB is considered to have a moderate reproductive toxicity by USEPA. In a 2-generation
oral gavage reproductive toxicity study in rats, no reproductive effects were identified at
doses up to 165 mg/kg bw/day. This study was done with the commercial mixture FMBZ-54,
containing EH-TBB and BEH-TEBP, with the larger constituent being EH-TBB. 165 mg/kg
bw/day was the highest dose tested with the mixture FMBZ-54 and was considered as the
NOAEL. No adverse effects were observed in reproductive performance and fertility. This
NOAEL falls within the range of Moderate hazard criteria set up by USEPA (MPI Research
2008a, USEPA 2015).
Developmental Toxicity
EH-TBB is considered to have a moderate developmental toxicity by USEPA. In a 2-
generation oral gavage reproductive toxicity study in rats, given 15, 50, or 165 mg/kg-day of
FMBZ-54, it was found that pups at birth had lower body weights. Body weights were also
lower throughout lactation, in both first and second generation offspring. In the first-
generation female, premating body weight was lower. At lactation day 21, spleen weights
were decreased in first generation male pups and both male and female pups in second
generation. A NOAEL of 50 mg/kg bw/day was identified from this study based on the effects
on body weight. This NOAEL falls within the range of Moderate hazard criteria. However, it
was not very clear which component or components of the commercial mixture had caused
the observed developmental effects. LOAEL was estimated to be 165 mg/kg bw/day based on
this study. (MPI Research 2008a, USEPA 2015).
In an unpublished prenatal study by MPI Research (2008b), rats were exposed to 0, 50, 100,
300 mg/kg bw/day FMBZ-54 mixture on gestation days 6-19. There was increased incidence
of dams with sparse hair in the abdomen, lower gestation body weight, and lower food
43
consumption during gestation at doses higher than or equal to 100 mg/kg bw/day. At 100
mg/kg bw/day, lower fetal weight was observed. Incidence of fused cervical vertebral neural
arches increased in fetuses at the highest dose. At highest dose, increased incidence of fetal
ossification variations, including additional ossification centres to the cervical vertebral neural
arches, incomplete ossified skull bones (jugal, parietal, and squamosal), and unossified
sternebrae were also observed. For maternal toxicity, a NOAEL of 50 mg/kg bw/day and a
LOAEL of 100 mg/kg bw/day was estimated based on the above-described effects. For
developmental toxicity, NOAEL of 50 mg/kg bw/day and LOAEL of 100 mg/kg bw/day were
estimated based on decreased fetal weight (2008b, USEPA 2015).
Neurotoxicity
EH-TBB is conservatively designated to have a moderate neurotoxicity by USEPA. There is
very limited experimental data available and no data available on neurotoxicity screening. In a
28-day sub-chronic oral toxicity study in rats treated with FM-550 in doses 0, 160, 400, 1000
mg/kg bw/day, no neurotoxic effects were reported. The NOAEL in this study was reported to
be 1000 mg/kg bw/day, which was the highest dose tested with FM-550 (Chemtura 2006).
Repeated Dose Effects
EH-TBB is considered to have a moderate repeated dose effects, designated by USEPA based
on the two developmental and prenatal study already described above (MPI Research 2008a,
2008b).
A 28-day sub-chronic oral toxicity study was performed in rats, treated with 0, 160, 400, 1000
mg/kg bw/day. Unspecified kidney effects were reported at 1000 mg/kg bw/day. No systemic
effects were observed at 160 mg/kg bw/day, and therefore a NOEL was estimated based on
this observation. Based on kidney effects, a LOAEL of 1000 mg/kg bw/day was estimated.
NOAEL was estimated at 400 mg/kg/day (Chemtura 2006).
3.3 BEH-TEBP
In a report published in August 2015, The USEPA has evaluated a number of novel and
emerging brominated flame retardants that has been used as alternatives for phased-out
PBDEs. Bis(2-ethyl-1hexyl)tetrabromophthalate (BEH-TEBP) with CAS Number 26040-51-7
was one of the brominated flame retardants evaluated.
44
In addition, BEH-TEBP is a pre-registration substance under REACH (ECHA 2016b). It has
been classified as low production volume (LPV) chemical, which implies that the import or
production volume is more than 10 tonnes but less than 1000 tonnes per year.
3.3.1 Hazard Identification for BEH-TEBP
Toxicokinetic information
Experimental data with a commercial mixture FM-550 showed that it was possible for BEH-
TEBP to be absorbed after oral exposure. BEH-TEBP was found in the tissues of exposed
dams after exposure to the commercial mixture, however, not in the pups even though
exposure has been from gestation to lactation (Patisaul et al. 2013).
In in vitro tests, mono(2-ethylhexyl)tetrabromophthalate (CAS number 61776-60-1) was
found to the primary metabolite. In rat or human subcellular fractions, no metabolites of
BEH-TEBP was found. This metabolite was formed by purified porcine carboxylesterase at a
rate of 1.08 mol/min mg/protein. No phase two metabolite of the primary metabolite was
found (Roberts et al. 2012). BEH-TEBP has not been evaluated in humans (USEPA 2015).
BEH-TEBP has been metabolized in vitro in hepatic subcellular fractions of fathead minnow,
common carp, snapping turtle and wild-type mice (Bearr et al. 2012). There was no data
available on toxicokinetic properties of the pure BEH-TEBP compound after oral, inhalation
or dermal exposure.
Hazard Identification
This FR was evaluated by USEPA to have low acute toxicity. However, it was evaluated to
have a moderate carcinogenicity, genotoxicity, reproductive toxicity, developmental toxicity,
neurotoxicity and repeated dose toxicity.
It was stated by the REACH registration applicant that conclusion cannot be drawn for
bioaccumulation potential in mammals based on the results of study.
45
3.3.2 Toxicity and Dose-Response Information for BEH-TEBP
For acute mammalian toxicity, the acute oral lethality was estimated to have an LD50 of larger
than or equal to 2000 mg/kg in rats, based on two studies (Bradford et al. 1996, Chemtura
2006). BEH-TEBP was estimated to have uncertain potential for carcinogenicity, based on
professional judgement and analogy with closely related chemicals. BEH-TEBP is considered
to have a moderate genotoxicity. In a chromosomal aberration test with human lymphocytes,
there was a weak positive result for the test material RC9927; CASRN 26040-51-7 (Purity of
BEH-TEBP > 95%) (ACC 2004). Two other in vitro chromosomal aberration assays were
performed using a component of a commercial mixture FM-550 containing BEH-TEBP,
which yielded negative results (Chemtura 2006). In an in vivo mouse micronucleus assay, it
was found that BEH-TEBP did not cause gene mutation in bacteria or chromosomal
aberration (ACC 2004).
A study submitted by a REACH registration applicant estimated an LD50 of >5000 mg/kg
bw. 5 female and 5 male rats were administered a single oral dose of 5000 mg/kg bw of BEH-
TEBP by gavage and observed for mortality and clinical signs for 14 days. No death occurred
to any animal. Body weight gain was normal and on day 15, there was no relevant necropsy
finding (ECHA 2016b).
Reproductive Toxicity
BEH-TEBP is considered to have a moderate reproductive toxicity by USEPA. In a 2-
generation oral gavage reproductive toxicity study in rats treated with 15, 50, or 165 mg/kg-
day FMBZ-54, no reproductive effects were identified at doses up to 165 mg/kg bw/day. 165
mg/kg bw/day was the highest dose tested and was considered as the NOAEL. No adverse
effects on reproductive performance or fertility in rats were observed. This NOAEL falls
within the range of Moderate hazard criteria (MPI Research 2008a, USEPA 2015).
In a 28-day repeated dose dietary toxicity study in rats given 0, 200, 2,000, and 20,000 ppm in
diet (approx. 0, 21.1, 211, 2,110 mg/kg bw/day) of test material RC9927; CASRN 26040-51-
7 (Purity of BEH-TEBP > 95%), no adverse changes in testes or ovary weights was observed.
Gross necropsy and histopathology were performed on a full complement of male and female
reproductive organs and tissues and no adverse effects were observed. However, other
46
reproductive indicators have not been examined. A NOAEL of 2000 ppm (approx. 223.4
mg/kg bw/day) for dietary toxicity was established. 2100 mg/kg bw/day was the highest dose
tested.
Developmental Toxicity
BEH-TEBP is considered to have a moderate developmental toxicity by USEPA. In a 2-
generation oral gavage reproductive toxicity study in rats, given 15, 50, or 165 mg/kg-day of
FMBZ-54, it was found that pups at birth had lower body weights. Body weights were also
lower throughout lactation, in both first and second generation offspring. In the first-
generation female, premating body weight was lower. At lactation day 21, spleen weights
were decreased in first generation male pups and both male and female pups in second
generation. A NOAEL of 50 mg/kg bw/day was identified from this study based on the effects
on body weight. This NOAEL falls within the range of Moderate hazard criteria. However, it
was not very clear which component or components of the commercial mixture had caused
the observed developmental effects. LOAEL was estimated to be 165 mg/kg bw/day based on
this study. (MPI Research 2008a, USEPA 2015).
In an unpublished prenatal study by MPI Research (2008b), rats were exposed to 0, 50, 100,
300 mg/kg bw/day FMBZ-54 mixture on gestation days 6-19. There was increased incidence
of dams with sparse hair in the abdomen, lower gestation body weight, and lower food
consumption during gestation at doses higher than or equal to 100 mg/kg bw/day. At 100
mg/kg bw/day, lower fetal weight was observed. Incidence of fused cervical vertebral neural
arches increased in fetuses at the highest dose. At highest dose, increased incidence of fetal
ossification variations, including additional ossification centres to the cervical vertebral neural
arches, incomplete ossified skull bones (jugal, parietal, and squamosal), and unossified
sternebrae were also observed. For maternal toxicity, a NOAEL of 50 mg/kg bw/day and a
LOAEL of 100 mg/kg bw/day was estimated based on the above-described effects. For
developmental toxicity, NOAEL of 50 mg/kg bw/day and LOAEL of 100 mg/kg bw/day were
estimated based on decreased fetal weight (2008b, USEPA 2015).
In a study submitted by REACH registration applicant, pregnant rats were administered BEH-
TEBP by oral gavage at dose levels of 250, 500 or 1000 mg/kg bw/day during gestation. No
effect was found on body weight development and dietary intake. A NOEL for maternal
47
toxicity was established at 1000 mg/kg bw/day at the highest dose tested. No relevant adverse
effects were observed in the offspring. The NOEL for developmental toxicity was therefore
1000 mg/kg bw/day at the highest dose tested (ECHA 2016b).
Neurotoxicity
BEH-TEBP is conservatively designated to have a moderate neurotoxicity by USEPA. There
is very limited experimental data available and no data available on neurotoxicity screening.
In a 28-day sub-chronic oral toxicity study in rats treated with FM-550 in doses 0, 160, 400,
1000 mg/kg bw/day, no neurotoxic effects were reported. The NOAEL in this study was
reported to be 1000 mg/kg bw/day, which was the highest dose tested with FM-550
(Chemtura 2006).
Repeated Dose Effects
BEH-TEBP is considered to have a moderate repeated dose effects. In a 28-day dietary
toxicity study, a small decrease of body weight, as well as decreased phosphorus and calcium
levels in female rats was observed. A LOAEL of 2110 mg/kg bw/day was estimated. A
NOAEL was identified as 211mg/kg bw/day. A moderate hazard was designated
conservatively (ACC 2004).
In a 2-generation oral reproductive toxicity study in rats, a NOAEL of 50 mg/kg bw/day was
estimated based on reduced body weight or body weight gain during premating period in
parental F0 and F1 female rats dosed with 165 mg/kg bw/day of the same commercial
mixture. LOAEL was determined to be 165 mg/kg bw/day (Chemtura 2006).
In a repeated-dose oral toxicity study submitted by REACH registration applicant, three
groups of ten male and ten female rats received BEH-TEBP (Tradename FR-45B) by diet at
200, 2000 or 20000 ppm concentrations (= approx. 21.97, 223.4 or 2331 mg/kg bw/day) for
four weeks. A similar control group received no treatment. A positive control group with five
males and five female rats received di-2-ethyl hexyl phthalate (DEHP) by diet at
concentration 15000 ppm for four weeks. Dietary administration of BEH-TEBP to rats at the
highest dose of 20000 ppm produced only minor changes, namely, there was a slightly lower
48
overall bodyweight gain for female rats. Marginally low alanine amino-transferase activities
were also observed in females receiving the highest dose. Marginally low phosphorus
concentrations were seen in all females and males receiving the highest dose. No evidence of
toxicity was observed at 200 or 2000 ppm. BEH-TEBP did not cause any toxicity in the liver
or testes observed in positive controls given DEHP, namely peroxisome proliferation. The
NOAEL was conservatively estimated at 2000 ppm, i.e. 223.4 mg/kg bw/day for the above-
described observed effect. This study and NOAEL has been used by the REACH registrant
and ECHA to estimate the DNEL for long-term oral exposure.
49
4 EXPOSURE ASSESSMENT
4.1 Available Relevant Exposure Information
Ali et al. (2011) estimated exposure to FRs via ingestion of indoor dust by assuming 100%
absorption of intake, as it was also done in a previous study by Jones-Otazo et al. 2005. The
amount of dust ingestion was assumed to be of an average 50 mg per day for toddlers and 20
mg per day for adults. For high dust ingestion, 200 mg per day was assumed for toddlers and
50 mg per day for adults. “Low”, “Typical” and “High” exposure scenarios for each
microenvironment were estimated using 5th percentile, median, and 95th percentile
concentrations in the dust samples. Overall exposure to FR by dust ingestion was calculated
according to the estimated relative time spent in each microenvironment for toddlers and
adults.
In the Ali study, in a typical exposure scenario, exposure to BTBPE was 0.01 ng/kg bw/day
for mean dust ingestion and 0.05 ng/kg bw/day for high dust ingestion for toddlers at the
median. For non-working adult, the typical exposure was 0.02 ng/kg bw/day for mean dust
ingestion and 0.06 ng/kg bw/day for high dust ingestion at the 95th percentile (below LOQ at
median). For working adult, the typical exposure was 0.04 ng/kg bw/day for mean dust
ingestion and 0.08 ng/kg bw/day for high dust ingestion at the 95th percentile (below LOQ at
median).
Exposure to EH-TBB was 0.02 ng/kg bw/day for mean dust ingestion and 0.08 ng/kg bw/day
for high dust ingestion for toddlers at the median. For non-working adult, the typical exposure
was 0.02 ng/kg bw/day for mean dust ingestion and 0.05 ng/kg bw/day for high dust ingestion
at the 95th percentile (below LOQ at median). For working adult, the typical exposure was
also 0.02 ng/kg bw/day for mean dust ingestion and 0.05 ng/kg bw/day for high dust ingestion
at the 95th percentile (below LOQ at median).
Exposure to BEH-TEBP was 0.10 ng/kg bw/day for mean dust ingestion and 0.40 ng/kg
bw/day for high dust ingestion for toddlers at the median. For non-working adult, the typical
exposure was 0.13 ng/kg bw/day for mean dust ingestion and 0.32 ng/kg bw/day for high dust
ingestion at the 95th percentile (below LOQ at median). For working adult, the typical
50
exposure was 0.01 ng/kg bw/day for mean dust ingestion and 0.02 ng/kg bw/day for high dust
ingestion at the median.
4.2 Brominated Flame Retardants Measured in Children’s Room in Kuopio, Finland
The Institute for Health and Welfare investigated the levels of novel and emerging flame
retardants in the indoor dust of 40 children’s room in the city of Kuopio, Finland. This study
has not been published. This section presents a part of the result from this study. The levels of
brominated flame retardants ab-DBE-DBCH, PBT, TBP-DBPE, EH-TBB, BTBPE, BEH-
TEBP will be presented in this section.
Indoor dust samples were collected through 2014-2015 with nylon socks by vacuum cleaner
from the floor of children’s rooms in 40 homes in and around the city of Kuopio, with an
average area of 8-10 m2. Dust samples were sieved through standard tea filters to remove
large particles and hair. Fifty mg of dust was weighed for each sample. Samples were
analysed with method described in section 2. Table 15 Shows the level of brominated flame
retardants found in children’s rooms in Kuopio.
Table 15. Levels of BFR found in children’s rooms in Kuopio (n=40).
Compound
DBE-
DBCH PBT
TBP-
DBPE
EH-
TBB
BTBP
E
BEH-
TEBP
Mean (ng/g) 0.554 1.135 1.464 6.030 2.722 242.715
Median 0.420 0.644 0.474 3.688 1.271 106.306
STDEV 0.410 2.263 3.418 5.818 4.071 354.452
5th Percentile 0.234 0.220 0.082 0.911 0.372 22.859
95th Percentile 1.241 1.851 5.914 17.913 6.708 887.262
Table 16 shows the levels of the above six BFRs detected in recent studies in homes
(bedrooms) in Boston, USA (Stapleton et al. 2008), Belgian homes (Ali et al. 2011), homes in
New Zealand (Ali et al. 2012), home in Romania (Dirtu et al. 2012), homes in Norway
(Cequier et al. 2014), homes in Durham, USA (Stapleton et al. 2014) and homes in Canada
(Fan et al. 2016). Not all six BFRs have been analysed in every study.
From these studies, it can be observed that the levels of EH-TBB seem comparable between
the THL study and from levels measured in New Zealand and Romanian homes. The levels of
51
BTBPE seem comparable across all studies. However, all studies show a much bigger range,
in particular those measured from homes in Boston, USA, Belgium and Canada. The levels of
BEH-TEBP from the THL study is comparable to those measured in bedrooms in Boston,
USA, Belgian homes, and Norwegian homes. The level of BEH-TEBP, as well as EH-TBB
measured from homes in Durham, USA appear to be considerably higher than that from the
THL study. The levels of FR measured in Canadian homes have a relatively wide range given
a much larger sample size of n=351.
52
Table 16. Concentrations (ng/g) of BFR in indoor dust in the THL study and those reported in other studies from Europe and USA. Results
expressed as median (range).
Reference Sample
Origin
Particle
Size (µm)
Sample
Mass (g)
Year of
Sampling
No. of
Samples (n)
DBE-
DBCH
PBT TBP-
DBPE
EH-TBB BTBPE BEH-
TEBP
THL Study Kuopio,
Finland
<500 0.05 2014-2015 40 0.42
(0.202-
2.17)
0.644
(0.140-
14.5)
0.474
(0.054-
19.4)
3.69
(0.586-
22.5)
1.27
(0.270-
24.9)
106 (12-
1930)
Stapleton et
al. 2008
Boston,
MA, USA
<500 ~0.3 2006 14 90.4*
(<10.6-
378)
47.8* (1.6-
789)
105* (1.5-
763)
Ali et al.
2011
Belgium <500 ~0.075 2010 39 1 (<2 –
436)
2 (<0.5-
1019)
13 (<20-
1286)
Ali et al.
2012
New
Zealand
<500 ~0.075 2008 34 2 (<2-
2285)
2 (<2-175) 12 (<2–
640)
Dirtu et al.
2012
Iasi,
Romania
<500 ~0.075 2010 47 <2 (<2-21) 4 (<2-90) 20
Cequier et al.
2014
Oslo,
Norway
<3000 >0.1 2012 48 ∼2 ng/g ∼3 ng/g ∼5 ng/g 78.5
Stapleton et
al. 2014
Durham,
NC, USA
<500 ~0.1 2009-2010 30 97 (6.0-
2430)
604 (82.9-
20960)
Fan et al.
2016
Canada <80 ~0.1 2007-2010 351 <0.6 (<0.6-
46)
Not
Detected
104 (<1.5-
13000)
8.5 (<1.7-
2390)
* Geometric mean
53
4.3 Exposure Estimation
This exposure estimation, shown below in Table 17, was based on the study by Ali et al.
(2011). Levels of BFR in indoor dust have been taken from the unpublished THL study
presented in section 4.2. In this exposure estimation, 100% absorption of intake is assumed, as
is done by Ali et al. (2011) and Jones-Otazo et al. (2005). In both aforementioned studies,
adult dust ingestion was assumed to be 20 mg/day for average ingestion, and 50 mg/day for
high ingestion. Toddler dust ingestion was assumed to be 50 mg/day for average ingestion,
and 200 mg/day for high ingestion.
Adult body weight was assumed to be 70kg, and toddler body weight assumed to weigh 10kg,
based on dose exposure calculation guidelines developed by the United States
Agency for Toxic Substances and Disease Registry (ATSDR 2005).
Table 18 presents a comparison of exposure values estimated from THL studies to studies by
Ali et al. (2011) estimated from Belgian homes & Ali et al. (2012) estimated from homes in
New Zealand. The exposure to EH-TBB and BTBPE are comparable across the three studies.
Toddler exposure to all three BFRs at 95th percentile in Belgian homes tend to be higher
compared to the other two studies. It is observed that exposure to BEH-TEBP from the THL
study, with samples taken from children’s rooms in Kuopio, is observed to be at least 2 times
higher than the other two studies for both toddler and adult exposure for all exposure ranges.
This may be due to a high median of 106 ng/g BEH-TEBP indoor dust level from the THL
study used for the exposure estimation. The median used for the Ali et al. studies were 13
ng/g for Belgian homes (2011) and 12 ng/g for New Zealand homes (2012). The range of
BEH-TEBP levels from the Ali et al. (2011) study of Belgian homes is comparable to that of
the THL study. The range from the 2012 study of New Zealand homes is smaller, as can be
seen in Table 16 above.
54
Table 17. Estimated oral exposure to BFRs through dust ingestion.
Toddler (10 kg bw) Adult (70 kg bw)
(ng/kg bw/day) 5th Percentile 95th Percentile Median 5th Percentile 95th Percentile Median
DBE-DBCH
Mean dust ingestion 0.001 0.006 0.002 0.000 0.000 0.000
High dust ingestion 0.005 0.025 0.008 0.000 0.001 0.000
PBT
Mean dust ingestion 0.001 0.009 0.003 0.000 0.001 0.000
High dust ingestion 0.004 0.037 0.013 0.000 0.001 0.000
TBP-DBPE
Mean dust ingestion 0.000 0.030 0.002 0.000 0.002 0.000
High dust ingestion 0.002 0.118 0.009 0.000 0.004 0.000
EH-TBB
Mean dust ingestion 0.005 0.090 0.018 0.000 0.005 0.001
High dust ingestion 0.018 0.358 0.074 0.001 0.013 0.003
BTBPE
Mean dust ingestion 0.002 0.034 0.006 0.000 0.002 0.000
High dust ingestion 0.007 0.134 0.025 0.000 0.005 0.001
BEH-TEBP
Mean dust ingestion 0.114 4.436 0.532 0.007 0.254 0.030
High dust ingestion 0.457 17.745 2.126 0.016 0.634 0.076
55
Table 18. Comparison of exposure values estimated from THL studies to studies by Ali et al. (2011 & 2012).
Toddler (10 kg bw) Adult (70 kg bw)
(ng/kg bw/day) 5th Percentile 95th Percentile Median 5th Percentile 95th Percentile Median
EH-TBB (Ali et al. 2011)
Mean dust ingestion 0.000 0.280 0.020 0.000 0.020 0.000
High dust ingestion 0.010 1.140 0.080 0.000 0.050 0.000
EH-TBB (Ali et al. 2012)
Mean dust ingestion 0.010 0.050 0.010 <0.01 <0.01 <0.01
High dust ingestion 0.020 0.200 0.040 <0.01 0.010 <0.01
EH-TBB (THL)
Mean dust ingestion 0.005 0.090 0.018 0.000 0.005 0.001
High dust ingestion 0.018 0.358 0.074 0.001 0.013 0.003
BTBPE (Ali et al. 2011)
Mean dust ingestion 0.000 0.350 0.010 0.000 0.020 0.000
High dust ingestion 0.000 1.390 0.050 0.000 0.060 0.000
BTBPE (Ali et al. 2012)
Mean dust ingestion <0.01 0.050 <0.01 <0.01 <0.01 <0.01
High dust ingestion 0.010 0.200 0.010 <0.01 0.010 <0.01
BTBPE (THL)
Mean dust ingestion 0.002 0.034 0.006 0.000 0.002 0.000
High dust ingestion 0.007 0.134 0.025 0.000 0.005 0.001
BEH-TEBP (Ali et al. 2011)
Mean dust ingestion 0.020 2.150 0.100 0.000 0.130 0.000
High dust ingestion 0.060 8.610 0.400 0.000 0.320 0.010
BEH-TEBP (Ali et al. 2012)
Mean dust ingestion <0.01 0.260 0.050 <0.01 <0.01 0.020
56
High dust ingestion 0.100 1.020 0.190 <0.01 0.040 0.010
BEH-TEBP (THL)
Mean dust ingestion 0.114 4.436 0.532 0.007 0.254 0.030
High dust ingestion 0.457 17.745 2.126 0.016 0.634 0.076
57
5 RISK CHARACTERISATION FOR BEH-TEBP
Based on the exposure estimation from Table 16 for BEH-TEBP, a Risk Characterisation
Ratio has been calculated based on the derived no-effect level (DNEL) of 0.37 mg/kg bw/day
(ECHA 2016b) for oral exposure in the general population. This DNEL has been derived for
long-term oral exposure according to a repeated dose oral toxicity study described in Section
3.3.2. The RCR calculated are shown in Table 18 below.
Table 18. RCR Calculation for BEH-TEBP based on DNEL of 0.37 mg/kg bw/day for oral
exposure in the general population. Toddler (10 kg bw) Adult (70 kg bw)
5th Percentile 95th Percentile Median 5th Percentile 95th Percentile Median
BEH-TEBP
Mean dust ingestion 0.0000003 0.0000120 0.0000014 0.0000000 0.0000007 0.0000001
High dust ingestion 0.0000012 0.0000480 0.0000057 0.0000000 0.0000017 0.0000002
6. DISCUSSION
Summary and Data Gaps for BTBPE
BTBPE is primarily eliminated in the faeces. It is shown that BTBPE has poor gastrointestinal
absorption in rats, however, a small percentage (<0.01%) of BTBPE may persist in the
adipose tissue, kidney, skin, and thymus. BTBPE has a high LD50 of >10g/kg bw. BTBPE
was found not mutagenic in Ames test and in yeast. There are also no hatching effects in
chicken. Based on available information, unspecified histopathological lesions in the lungs
has been observed in rats after inhalation exposure for 21 days. Based on this limited
information, inhalation exposure in humans may be important in terms of possible health risk.
However, there is no information available for BTBPE on any human health endpoints.
For BTBPE, there is very limited published information regarding human health risk and
human health end-points. Therefore, information on carcinogenicity, reproductive toxicity,
developmental toxicity, neurotoxicity and repeated-dose toxicity are not available.
Exposure to BTBPE is among the lowest in the list of BFR considered, however, toddler
exposure to BTBPE for high dust ingestion in the 95th percentile can exceed 1 ng/kg bw/day.
58
Even though BTBPE is considered a LPV chemical, more information on human health-
related end-points should be made available.
Summary and Data Gaps for EH-TBB
Toxicokinetic study was conducted with the commercial mixture FM-550 containing a
mixture of EH-TBB and BEH-TEBP. Absorption was possible through oral exposure from
gestation and through lactation in rats. EH-TBB and BEH-TEBP were detected in adipose,
liver and muscle tissues of dams, and in pup adipose tissue on post-natal day 21. The primary
metabolite of EH-TBB was found in the liver tissue of dams on post-natal day 21. EH-TBB
has an LD50 of >5000 mg/kg bw. The commercial mixture FMBZ-54 (a mixture of EH-TBB
and BEH-TEBP) was found to have no adverse effect in reproductive performance and
fertility at 165 mg/kg bw/day. However, in a prenatal study with rats, it was found that there
was higher incidence of dams with sparse hair in the abdomen, lower gestation body weight,
and lower food consumption during gestation at dose 100 mg/kg bw/day. At this dose level,
lowered fetal weight was observed. At 300 mg/kg bw/day, there was increased incidence of
fused cervical vertebral neural arches in fetus, and other developmental effects. With the
consideration of human health, it is significant that absorption of EH-TBB was found to be
possible through lactation. Deposition of EH-TBB was also possible in adipose, liver and
muscle tissues. Moreover, at high dose, adverse effects were observed in pregnant rat dams
and in rat foetal development. Therefore, foetal exposure and neonatal exposure through
lactation, as well as accumulation in adipose can be a human health concern.
There was only one study reported for neurotoxicity with no effect observed at 1000 mg/kg
bw/day. Apart from this, there is very limited experimental data available and no data
available on neurotoxicity screening. Details on kidney effects from the 28-day sub-chronic
oral toxicity study performed by Chemtura (2006) has not be made publicly available.
Exposure to EH-TBB is among the lowest in the list of BFR considered, however, toddler
exposure to EH-TBB for high dust ingestion in the 95th percentile can exceed 1 ng/kg
bw/day. Given the above possible human health concern, especially in feotal development,
neurotoxicity studies would be informative. Human biomonitoring of breast milk would also
be relevant.
59
Summary and Data Gaps for BEH-TEBP
Toxicokinetic study was conducted with the commercial mixture FM-550. BEH-TEBP was
found in the tissues of exposed dams after exposure to the commercial mixture, however, not
in the pups even though exposure has been from gestation to lactation. BEH-TEBP has an
LD50 of >5000 mg/kg bw. The REACH registrant for BEH-TEBP has provided studies
conducted with the compound BEH-TEBP of >95% purity. This test material was found to
cause no adverse reproductive effects in rats, with an estimated NOAEL of approximately
223.4 mg/kg bw/day dietary exposure. No developmental effects were observed at 1000
mg/kg bw/day, which was the highest dose tested and the NOEL. For repeated-dose oral
toxicity, NOAEL was conservatively estimated at 2000 ppm, i.e. 223.4 mg/kg bw/day for
minor observed effects. There was a slightly lower overall bodyweight gain for female rats.
Marginally low alanine amino-transferase activities were also observed in females receiving
the highest dose of 2331 mg/kg bw/day Marginally low phosphorus concentrations were seen
in all females and males receiving the highest dose. This study and the estimated NOAEL has
been used by the REACH registrant and ECHA to estimate the DNEL for long-term oral
exposure.
For BEH-TEBP, there was only one study reported for neurotoxicity with no effect observed
at 1000 mg/kg bw/day. Apart from this, there is very limited experimental data available and
no data available on neurotoxicity screening. Neurotoxicity study has not been included in the
registration dossier.
Among international results as well as results from THL (Table 16), BEH-TEBP was present
at the highest level among the six BFR measured. The estimated exposure was also highest
(Table 17, Table 18). Based on the THL study, the estimated exposure at median
concentration for mean dust ingestion was 0.532 ng/kg bw/day for toddlers and 0.030 ng/kg
bw/day for adults. However, for high dust ingestion at 95th percentile concentration, the
exposure can reach 17.7 ng/kg bw/day for toddlers and 0.634 ng/kg bw/day for adults. BEH-
TEBP should be of particular concern due to the relatively high level of exposure. Therefore,
information on neurotoxicity would be relevant. Human biomonitoring of serum and breast
milk concentration of BEH-TEBP would also be relevant.
60
Discussion on Risk Characterisation
Based on the DNEL of 0.37 mg/kg bw/day for oral exposure in the general population, the
RCR calculated for all exposure scenarios for both toddlers and adults are well below 1,
indicating that the risk from oral exposure through house dust is adequately controlled.
However, it must be kept in mind that the sample size of 40 from children’s rooms in Kuopio,
Finland is insufficient to represent the Finnish population, nor the population of the city of
Kuopio. Moreover, RCR has been calculated based on the exposure estimation through house
dust alone, combined exposure has not been considered.
The DNEL employed in the risk characterization is considered reliable. Toxicological
information from the REACH registration dossier for BEH-TEBP are based on study with
high reliability, with a denomination of 1 – reliable without restrictions, or 2 – reliable with
restrictions.
Further Considerations
Many of the toxicological studies have been conducted with commercial mixtures of EH-TBB
and BEH-TEBP, namely FM-550 and FMBZ-54. In the REACH registration dossier for BEH-
TEBP, tests have been conducted with the compound BEH-TEBP of >95% purity. This test
material was found to cause no adverse reproductive effects in rats at 223.4 mg/kg bw/day
dietary exposure for four weeks, and no developmental effects were observed at 1000 mg/kg
bw/day when exposed during gestation. However, the commercial mixture FMBZ-54 was
found to cause maternal toxicity at 100 mg/kg bw/day and foetal developmental effects at
300 mg/kg bw/day when exposed during gestation day 6-19.
It is reasonable to assume that since these commercial mixtures are being applied to furniture,
humans would be exposed to flame retardants as mixtures as well. Moreover, in many studies
conducted with commercial mixtures, it is unclear which components of the mixture may be
driving the observed adverse effects (USEPA 2015). Therefore, it is important to consider
human exposure to flame retardants as mixtures, and the possibility of synergistic effect of
these chemical mixtures. Further studies in this respect would be very informative.
61
7. SUMMARY AND CONCLUSIONS
Flame retardants (FR) have been added to a wide range of industrial and commercial products
as a result of flammability standards, therefore FRs are ubiquitous in the indoor environment.
FRs can accumulate in indoor dust, and levels of FRs present in house dust can be an
indication of human exposure to FRs. Brominated flame retardants (BFR) and
Organophosphorous Flame Retardants (OPFR) have been developed to replace banned
Polybrominated diphenyl ethers (PBDE). However, the toxicity of these novel and emerging
FRs to human health, and possible tendency for bio-accumulation have become a concern.
Method development and validation for analysis of flame retardants in house dust that was
performed between May-August 2015 based on the method developed by Van den Eede et al.
2012. The principle aim of this method development is to develop and validate an effective
analysis method for indoor dust containing selected novel and emerging BFR, OPFR, as well
as PBDEs. In the second part of this thesis, a human health risk assessment for one of the
above selected BFRs, namely BEH-TEBP was performed based on currently available
scientific literature and estimated exposure from dust ingestion.
There was insufficient toxicological and exposure information available for ab-DBE-DBCH,
PBT, TBP-DBPE to perform a hazard identification. Relevant information available for the
substances BTBPE, EH-TBB and BEH-TEBP were summarized in Section 3. An exposure
estimation has been performed based on the amount of BFR detected from a recent
unpublished study by THL. Based on this exposure estimation, Risk Characterisation Ratio
(RCR) has been calculated for BEH-TEBP according to the derived no-effect level (DNEL)
for oral exposure.
Dichloromethane was chosen to be the most suitable extraction solvent due to ease of
preparation and use. Direct pouring of 4ml Dcm extract to cleanup columns was chosen for
future implementation. It was also concluded that 100% Dcm was the most suitable solvent
for the elution of Fraction 1 from the dual clean-up columns with Florisil column on top of
H2SO4-silica column was used for Fraction 1 only. With this solvent most of the BFRs could
be separated from the OPFRs. 10% Acetone-Dcm was determined to be the most suitable
solvent for the elution of Fraction 2, where all of OPFR, could be eluted, except that 50% of
62
TBOEP still remained in the column. BEH-TEBP was eluted in Fraction 2 together with
OPFR. Settled dust was considered to be a preferable matrix.
For the BFRs, percentage recovery of internal standards were found to be in the range of 60%
- 120%. Ion ratios of compounds to be measured were within +/- 20% of the theoretical
values, or values from calibration standards. For the OPFRs, chromatography peak sizes
tended to be too small in the calibration standard for all compounds, and for some compounds
in the Internal Standard solution added to samples.
For the validation, Limit of Quantification (LOQ) & Measurement Uncertainties were
acceptable for all BFRs. LOQ of BFRs ranged between 0.5 – 5.0 ng/g. For OPFR, LOQ
ranged between 6.9-613 ng/g. Precision for OPFR was good, but accuracy was poor compared
to other labs’ analysis result for the certified material for indoor dust SRM 2585. Therefore,
method for OPFR analysis requires further testing that was not conducted within the scope of
this thesis. Exposure estimation was based on the study by Ali et al. (2011), where 100%
absorption of intake is assumed. Adult dust ingestion was assumed to be 20 mg/day for
average ingestion, and 50 mg/day for high ingestion. Toddler dust ingestion was assumed to
be 50 mg/day for average ingestion, and 200 mg/day for high ingestion.
For the purpose of estimating exposure, unpublished results from the National Institute for
Health and Welfare (THL) were used. The detected amount of BEH-TEBP from children’s
room in Kuopio, Finland (n=40) had a median of 106.3 ng/g, with a range of 22.8 ng/g –
887.2 ng/g (5th to 95th percentile). A Risk Charactrisation Ratio has been calculated based on
the DNEL of 0.37 mg/kg bw/day (ECHA 2016b) for oral exposure in the general population.
All RCR derived for given exposure scenarios are less than 1, meaning that the risk is
adequately controlled. However, it must be kept in mind that combined exposure has not been
taken into consideration.
63
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67
APPENDICES
APPENDIX 1: Reference values for BFR and OPFR analysis in house dust
Compoun
d (Abbr.)
CAS-
Numbe
r
Full Chemical name Certifie
d Value
(SRM-2585)
ng/g
Ali 2011
(ng/g) -
Indicative Value
Van den
Eede 2011
(ng/g) - Indicative
Value
Van den
Eede 2012
(ng/g) - Measured
Value
Sahlströ
m 2012
(ng/g)
Cristale
2013
(ng/g)
Stapleto
n 2008
(ng/g)
Hoffma
n 2015
(ng/g)
Bergh 2012
(ng/g)
Fan
2016
(ng/g)
Iona
s
2013 (ng/g
)
Fromm
e 2014
(ng/g)
Ali 2012
(ng/g)
TCEP 115-
96-8
Tris(2-chloroethyl)
phosphate
700(170) 680(60) 840(60) 700 788(21)
TCIPP 13674-
84-5
Tris(1-chloropropan-2-yl)
phosphate
820(100) 860(70) 880(140) 1000 840(19)
TDCIPP 13674-
87-8
tris(1,3-
dichloroisopropyl) phosphate
2020(260) 3180(70) 1820(9
0)
2300(280) 2200 1936(95)
TDBPP 126-
72-7
Tris(2,3-dibromopropyl)
phosphate
TIBP 126-
71-6
Tri(isobutyl)phosphate <(MDL=29
0)
300 1565(388)
TNBP 126-
73-8
Tri-n-butylphosphate 180(20) 190(10) 190(20) 400 169(20)
TBOEP 78-51-
3
Tris(2-
butoxyethyl)phosphate
49,000(960
0)
63,000(200
0)
82000(6500
), 73000
71000 40,107(32
4)
EHDPP 1241-
94-7
2-Ethylhexyl diphenyl
phosphate
1300(120),
1000
1900
TEHP 78-42-
2
Tris(2-
ethylhexyl)phosphate
370(40),
330
500
TPHP 115-
86-6
Triphenyl phosphate 990(70) 1160(140) 520
(34)
1100(100) 700 1,058(85)
TMPP 1330-
78-5
Tris (methylphenyl)
phosphate
1070(110) 1140(30) 740(110)
ab-DBE-DBCH
3322-93-8
alpha/beta-Tetrabromoethylcyclohex
ane
PBT 87-83-
2
Pentabromotoluene
PBEB 7.7(0.6)
HBB 2.8(0.4) 2(0.8
)
TBP-
DBPE
35109-
60-5
2,3-Dibromopropyl 2,4,6-
tribromophenyl ether
68
EH-TBB 18365
8-27-7
2-Ethylhexyl-2,3,4,5-
tetrabromobenzoate
40 26(2) 36(2.4) 35(6) 38.8(4.
8)
40(2
)
BTBPE 37853-59-1
1,2-Bis(2,4,6-tribromophenoxy)ethane
32 39(14) 39(4.9) 76(4) 37.8(5.9)
35(4)
BEH-
TEBP
26040-
51-7
Bis(2-ethyl-
1hexyl)tetrabromophthala
te
652 574(49) 1300(94
)
857(73) 145(16.
7)
DBDPE 84852-53-9
Decabromodiphenylethane
< 20 <7.1
BDE-28 2,2',4-Tribromodiphenyl
Ether
46.9
(4.4)
32.8(1.1) 42(1)
BDE-47 2,2',4,4'-Tetrabromodiphenyl
Ether
497 (46)
409(11) 486(10)
BDE-99 2,2',4,4',5-
Pentabromodiphenyl Ether
892
(53)
742(23) 803(45)
BDE-100 2,2',4,4',6-
Pentabromodiphenyl
Ether
145
(11)
116(3) 147(5)
BDE-153 2,2',4,4',5,5'-
Hexabromodiphenyl
Ether
119 (1) 97(2) 118(8)
BDE-154 2,2',4,4',5,6'-
Hexabromodiphenyl Ether
83.5
(2.0)
77.2(2.7) 77(5)
BDE-183 2,2',3,4,4',5',6-
Heptabromodiphenyl
Ether
43.0
(3.5)
32.3(4.8) 44(4)
BDE-209 Decabromodiphenyl Ether 2510
(190)
2150(231) 2971(33
3)
* (ng/g) = (µg/kg) [a] Ali, N. et al. 2011. ‘Novel’’ brominated flame retardants in Belgian and UK indoor dust: Implications for human exposure. Chemosphere 83 (2011) 1360-1365.
[b] Cristale,J.; Lacorte,S. 2013. Development and validation of a multiresidue method for the analysis of polybrominated diphenyl ethers, new brominated and organophosphorus flame retardants in sediment, sludge and
dust. J. Chromatogr. A. 1305 (2013) 267-275.
[c] Sahlström et al. 2012. Clean-up method for determination of established and emerging brominated flame retardants in dust. Anal. Bioana. Chem. 404 (2912) 459.
[d] Stapleton H.M. et al. 2008. Alternate and new brominated flame retardants detected in U.S. house dust. Enviro. Sci. Technol. 42 (2008) 6910. [e] Van den Eede, N. et al. 2011. Analytical developments and preliminary assessment of human exposure to organophosphate flame retardants from indoor dust. Environment International 37(2) 454-461.
[f] Van den Eede, N. et al. 2012. Multi-residue method for the determination of brominated and organophosphate flame retardants in indoor dust. Talanta 89 (2012) 292-300.
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69
APPENDIX 2: Criteria Used by USEPA to Assign Hazard Designation
Endpoint Very High High Moderate Low Very Low
Human Health Effects
Acute mammalian toxicity
Oral median lethal
dose (LD50) (mg/kg) ≤50
>50–300 >300–2,000
>2,000
–
Dermal LD50 (mg/kg) ≤200 >200–1,000 >1,000–2,000 >2,000 –
Inhalation median
lethal concentration
(LC50) - vapor/gas
(mg/L)
≤2 >2–10 >10–20 >20 –
Inhalation LC50 -
dust/mist/ fume
(mg/L)
≤0.5 >0.5–1.0
>1–5
>5
–
70
Carcinogenicity
Carcinogenicity
Known or presumed
human carcinogen
(equivalent to Globally Harmonized
System of
Classification and
Labeling of Chemicals (GHS)
Categories 1A and
1B)
Suspected human
carcinogen
(equivalent to GHS
Category 2)
Limited or marginal
evidence of
carcinogenicity in
animals
(and inadequate
evidence in humans)
Negative studies or
robust mechanism-
based Structure
Activity Relationship
(SAR)
(as described above)
–
71
Mutagenicity/Genotoxicity
Germ cell
mutagenicity
GHS Category 1A or
1B: Substances
known to induce
heritable mutations or
to be regarded as if
they induce heritable mutations in the germ
cells of humans
GHS Category 2:
Substances which cause concern for
humans owing to the
possibility that they
may induce heritable mutations in the germ
cells of humans
OR
Evidence of mutagenicity
supported by positive
results in in vitro OR
in vivo somatic cells
of humans or animals
Negative for
chromosomal
aberrations and gene
mutations, or no
structural alerts.
--
Mutagenicity and
genotoxicity in
somatic cells
Evidence of mutagenicity
supported by positive
results in in vitro
AND in vivo somatic
cells and/or germ
cells of humans or
animals
72
Reproductive toxicity
Oral (mg/kg/day) –
<50 50–250
>250-1,000
>1,000
Dermal (mg/kg/day) – <100 100–500 >500-2,000 >2,000
Inhalation - vapor,
gas (mg/L/day) – <1 1–2.5 >2.5-20 >20
Inhalation -
dust/mist/fume
(mg/L/day)
– <0.1 0.1–0.5 >0.5-5 >5
Developmental toxicity
Oral (mg/kg/day) – <50 50–250 >250-1,000 >1,000
Dermal (mg/kg/day) – <100 100–500 >500-2,000 >2,000
Inhalation - vapor,
gas (mg/L/day) – <1 1–2.5 >2.5-20 >20
Inhalation -
dust/mist/fume
(mg/L/day)
–
<0.1 0.1–0.5 >0.5-5 >5
73
Neurotoxicity
Oral (mg/kg/day) –
<10 10–100
>100
–
Dermal (mg/kg/day) – <20 20–200 >200 –
Inhalation - vapor,
gas (mg/L/day) - <0.2 0.2–1.0 >1.0 -
Inhalation -
dust/mist/fume
(mg/L/day)
- <0.02 0.02–0.2 >0.2 -
74
Repeated-dose toxicity
Oral (mg/kg/day) –
<10 10–100
>100
–
Dermal (mg/kg/day) – <20 20–200 >200 –
Inhalation - vapor, gas
(mg/L/day) - <0.2 0.2–1.0 >1.0 -
Inhalation - dust/mist/fume
(mg/L/day) - <0.02 0.02–0.2 >0.2 -
*Very High or Very Low designations (if an option for a given endpoint in Table 5-2) were assigned only when there were experimental data
located for the chemical under evaluation. In addition, the experimental data must have been collected from a well conducted study specifically
designed to evaluate the endpoint under review. If the endpoint was estimated using experimental data from a close structural analog, by
professional judgment, or from a computerized model, then the next-level designation was assigned (e.g., use of data from a structural analog
that would yield a designation of very high would result in a designation of high for the chemical in review). One exception is for the estimated
persistence of polymers with an average MW >1,000 daltons, which may result in a Very High designation.
** The details as to how each endpoint was evaluated are described below and in the DfE full criteria document, DfE Alternatives Assessment
Criteria for Hazard Evaluation, available at: http://www2.epa.gov/saferchoice/alternatives-assessment-criteria-hazard-evaluation.