Technical report: Silanamine, 1,1,1-trimethyl-N

Technical report: Silanamine, 1,1,1-trimethyl-N-(trimethylsilyl)-, hydrolysis products with silica; pyrogenic, synthetic amorphous, nano, surface treated silicon dioxide

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Agency technical report on the classification and labelling of

[Silanamine, 1,1,1-trimethyl-N-(trimethylsilyl)-, hydrolysis products with

silica; pyrogenic, synthetic amorphous, nano, surface treated silicon dioxide]

EC Number: 272-697-1 CAS Number: 68909-20-6

Health and Safety Executive Chemicals Regulation Division

Redgrave Court Merton Road

Bootle L20 7HS [email protected]

Date: June 2021

mailto:[email protected]


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Contents Brief summary ........................................................................................................................... 3

Introduction ............................................................................................................................... 4

Overview of current and proposed classification and labelling ................................................ 4

General substance information: ................................................................................................ 7

Background ................................................................................................................................ 7

Scientific assessment of the physical, human health and environmental hazard classes ........ 8

Physical Hazards ........................................................................................................................ 8

Health Hazards .......................................................................................................................... 8

Acute toxicity ......................................................................................................................... 8

Specific target organ toxicity – single exposure (STOT SE).................................................. 11

Skin corrosion/irritation ...................................................................................................... 12

Serious eye damage/irritation ............................................................................................. 12

Respiratory sensitisation ..................................................................................................... 13

Skin sensitisation ................................................................................................................. 13

Specific target organ toxicity – repeated exposure (STOT RE) ............................................ 14

Germ cell mutagenicity ....................................................................................................... 16

Carcinogenicity .................................................................................................................... 18

Reproductive toxicity ........................................................................................................... 18

Aspiration hazard ................................................................................................................ 19

Environmental hazards ............................................................................................................ 19

Hazardous to the aquatic environment .............................................................................. 19

Other hazards .......................................................................................................................... 24

Hazardous to the ozone layer ............................................................................................. 24

Overall conclusion ................................................................................................................... 25

References ............................................................................................................................... 26

Glossary of terms used in Agency technical reports ............................................................... 27


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Brief summary

The conclusion of the Agency technical report is that silanamine, 1,1,1-trimethyl-N-(trimethylsilyl)-, hydrolysis products with silica; pyrogenic, synthetic amorphous, nano, surface treated silicon dioxide meets the classification criteria for:

Acute Tox 2; H330 (Fatal if inhaled) with an ATE of 0.45 mg/L (dusts or mists) STOT RE 2 (lung; inhalation); H373 (May cause damage to lungs through prolonged or repeated exposure via inhalation) NOT CLASSIFIED for physical hazards, acute toxicity (via the oral and dermal routes of exposure), STOT SE, skin corrosion/irritation, serious eye damage/irritation, skin sensitisation, germ cell mutagenicity, carcinogenicity, reproductive toxicity and hazardous to the aquatic environment. The additional label EUH066 (Repeated exposure may cause skin dryness or cracking) should be applied. Is this in agreement with the RAC opinion? YES

At the time of publication, this mandatory classification and labelling has not been agreed and/or adopted in Great Britain. The following hazard classes are not assessed in this technical report: respiratory sensitisation, aspiration hazard and hazardous to the ozone layer. These hazard classes were not assessed in the CLH report or RAC opinion.


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Introduction

Under Article 37 of the GB CLP Regulation1, the Agency2 is required to produce a technical report for each substance on which the Committee for Risk Assessment (RAC) of the European Chemicals Agency produces an opinion3. This technical report documents an independent scientific assessment, conducted by HSE technical specialists with support from the Environment Agency for the environmental hazard classification, of the classification and labelling of silanamine. Table 1. Information considered in the scientific assessment

Document Included in assessment

EU CLH report Yes

Annexes to the EU CLH report Yes

RAC opinion Yes

Background document Yes

Information submitted during the EU public consultation process (RCOM table, including attachments)

Yes

RAC minority opinion(s) Yes

Other information: No

This information has been evaluated against the classification and labelling criteria set out in the GB CLP Regulation.

1The retained CLP Regulation (EU) No. 1272/2008 as amended for Great Britain 2 HSE acting in its capacity as the GB CLP Agency 3 Under Article 37(4) of Regulation (EU) No 1272/2008 on classification, labelling and packaging of substances and mixtures


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Overview of current and proposed classification and labelling

Table 2. Current and proposed classification and labelling

Classification

Labelling

Index No.

International Chemical

Identification

EC No. CAS No.

Hazard Class and Category

Code(s)

Hazard Statement

Code(s)

Pictogram, Signal Word

Code(s)

Hazard Statement

Code(s)

Suppl. Hazard

Statement Code(s)

Specific Concentration

Limits, M- factors

Notes

GB MCL List entry

No current entry

EU dossier submitter’s proposal

TBD silanamine, 1,1,1-

trimethyl-N- (trimethylsilyl)-,

hydrolysis products with

silica; pyrogenic, synthetic

amorphous, nano, surface treated silicon

dioxide

272- 697-1

68909- 20-6

STOT RE 2 H373 (lungs,

inhalation)

GHS08 Wng

H373 (lungs,

inhalation)

EUH 066

EU RAC opinion




silica; pyrogenic,

272- 697-1

68909- 20-6

Acute Tox. 2 STOT RE 2

H330 H373

(lungs, inhalation)

GHS06 GHS08

Dgr

H330 H373

(lungs, inhalation)

EUH066 ATE = 0.45 mg/L (dusts or

mists)


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Classification

Labelling

Index No.


Identification

EC No. CAS No.


Code(s)

Hazard Statement

Code(s)


Code(s)

Hazard Statement

Code(s)

Suppl. Hazard

Statement Code(s)


Limits, M- factors

Notes

synthetic amorphous,

nano, surface treated silicon

dioxide

Agency technical report conclusion






dioxide

272- 697-1

68909- 20-6


H330 H373 (lungs, inhalation)

GHS06 GHS08 Dgr


EUH066 ATE = 0.45 mg/L (dusts or mists)

Resulting MCL entry on GB MCL list





272- 697-1

68909- 20-6



GHS06 GHS08 Dgr


EUH066 ATE = 0.45 mg/L (dusts or mists)


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Classification

Labelling

Index No.


Identification

EC No. CAS No.


Code(s)

Hazard Statement

Code(s)


Code(s)

Hazard Statement

Code(s)

Suppl. Hazard

Statement Code(s)


Limits, M- factors

Notes


dioxide

TBD: to be determined


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General substance information:

Active substance in Plant Protection Products: ☐

Active substance in Biocidal Products: ☒

Chemical registered under REACH: ☐

Background

Silanamine, 1,1,1-trimethyl-N-(trimethylsilyl)-, hydrolysis products with silica; pyrogenic, synthetic amorphous, nano, surface treated silicon dioxide (hereinafter referred to as silanamine) is an active substance in the meaning of Regulation (EU) 528/2012 used in biocidal products. It is a hydrophobic synthetic amorphous silica (SAS) modified with hexamethyldisilazane (HMDS; trade name Aerosil R 812) to be used by professional operators in the control of fowl-infesting ectoparasites in poultry houses (product type 18). It falls under the general description of an engineered nanomaterial. The substance is considered inorganic with more than 95% of the hydrophobic amorphous silica comprised of polymerically bound silicon dioxide (SiO2) with minimal carbon content. The resulting solid particles are considered highly hydrophobic, very stable, insoluble in water, largely inert and non-volatile. They act by abrasion of the surface cuticle of susceptible arthropod parasites, so increasing their moisture loss and desiccation. Silanamine does not have an existing entry on the GB mandatory classification and labelling list or Annex VI of EU CLP. Since it is an active substance for use in biocidal products the EU dossier submitter (DS: France) submitted a CLH dossier that addressed all hazard end-points, in accordance with Article 36 of the CLP Regulation. The DS read-across data from the structurally related, hydrophilic substance Aerosil R 972 to supplement the information on silanamine for some effects. RAC made the following points in deciding not to incorporate the information on Aerosil R 972 in its assessment: significant differences in chemical structure and physicochemical parameters; hydrophilic and hydrophobic SAS differences in biological and environmental reactivity; lack of relevant data to support and justify possible read-across between the hydrophobic and hydrophilic forms of SAS. However, RAC concluded that SAS dimethyldichlorosilane (DDS) and SAS polydimethylsiloxane (PDMS) are sufficiently similar surface-modified SAS compared with silanamine and used these as source substances in the read-across assessment in the RAC opinion. RAC used the aforementioned Aerosil R 972 to provide further information on the toxicological profile of silanamine.


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Scientific assessment of the physical, human health and environmental hazard classes

Physical Hazards

Classification agreed by RAC: Silanamine is an inorganic, inert solid with mineral character that is almost fully oxidised with a high melting point so has no structural potential for explosive, flammable, self-reactive, pyrophoric, self-heating or oxidising properties. Therefore, RAC agreed with the DS that classification for physical hazards was not warranted for silanamine.

Classification proposed by the Agency: The Agency agrees that silanamine does not meet the criteria for classification for physical hazards.

Health Hazards

Acute toxicity

Classification agreed by RAC: Oral The DS referred to one GLP-compliant acute oral toxicity study (OECD 401) in rats in the CLH dossier, in which a single dose of 2000 mg/kg bw/d silanamine was administered. With an LD50 of >2000 mg/kg bw (the boundary for classification) in the available guideline study, silanamine did not meet the minimum criteria for Acute Tox. 4. RAC also referred to data from the hydrophobic surface-treated SAS-DDS and SAS-PDMS. RAC concluded that classification for acute oral toxicity was not warranted. Dermal An acute dermal toxicity study was not provided, but the DS referred to information from a skin irritation study with silanamine in which there were no deaths following administration of 0.5 g per animal (approximately 200 mg/kg bw/d). RAC read-across information from an acute dermal toxicity study with SAS-DDS, in which the LD50 value was >2000 mg/kg bw. The skin irritation study with silanamine provided supportive information.


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With an LD50 of >2000 mg/kg bw (the boundary for classification) in the available read-across study in SAS-DDS, silanamine did not meet the minimum criteria for Acute Tox. 4 and, therefore, RAC concluded that classification for acute dermal toxicity was not warranted. Inhalation The DS referred to two studies in the CLH report. In one GLP-compliant, non-guideline study with SAS-DDS in rats there were no deaths, pathological abnormalities or severe clinical signs of toxicity. The 4-hour LC50 value was >0.48 mg/L, which was the maximum attainable concentration. This concentration is below the recommended dose in OECD 403 and was only 2% of the nominal concentration (24400 mg/m3) because the electrostatic charge of the test substance particles caused large amounts of the test material to be deposited on the walls of the apparatus. The second test was a mechanistic study of a single intratracheal application of silanamine in rats, investigating lung-toxicity and DNA damaging effect. Evidence from an increased rate of neutrophils, the number of cells and an increase in protein content in rinsing solution indicated dose-dependent inflammation in the deep lung that was fully reversible within 21 days for all concentrations. RAC also considered literature studies using various forms of hydrophobic SAS, one of which, an OECD TG 403 test of SAS-DDS in rats, was considered a key study. After exposure to the test substance with a particle size of 0.8-1µm, a 4-hour LC50 value of 0.45 mg/L was found. RAC considered that the reduction in particle size might not necessarily reflect realistic conditions for silanamine and other hydrophobic SAS, but nevertheless believed that the intrinsic size of the substances was the nanoform and not the agglomerate and, therefore, they were considered to be nanomaterials. RAC proposed an ATE value of 0.45 mg/L. No comments were initially submitted regarding acute inhalation toxicity; however, as the studies leading to RAC’s decision on classification were not included in the CLH report, an ad hoc consultation was launched in February 2020. Thirteen comments from eight industries and five from individuals were received, highlighting scientific issues such as: the majority of studies were carried out before the release of OECD 403 guidelines so were not in compliance with current standards; reliability scores for studies were reviewed by independent experts and were downgraded; industry concluded that tests had no relevance to human exposure as particle size distribution in test substances was significantly reduced compared with the particle size of commercially used SAS; the tendency of SASs to agglomerate led industry to conclude lethality was due to suffocation rather than an intrinsic property of silanamine and, therefore, did not represent real-life conditions. In accordance with the CLP criteria, classification in Category 2 is permitted when an LC50 value >0.05 mg/L and ≤0.5 mg/L is observed in guideline studies. As this was


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detected in one of the key literature studies, RAC concluded that silanamine met this criterion. RAC therefore disagreed with the DS and concluded that classification for acute oral toxicity in category 2 (H330: Fatal if inhaled) was warranted. Classification proposed by the Agency: Oral The Agency agrees with RAC’s assessment of the data. Silanamine does not meet the criteria for classification for acute oral toxicity. Dermal The Agency agrees with RAC’s assessment of the data. Silanamine does not meet the criteria for classification for acute dermal toxicity. Inhalation The Agency notes the following factors. As stated in the background document, the surface-treated hydrophobic SAS (silanamine, SAS-DDS, SAS-PMDS) have a water solubility (saturation) of 100-160 mg/L at 37֯C and pH 7.1-7.4 (i.e., only slightly soluble). RAC noted that hydrophobicity can influence agglomeration and sorption, as well as dispersibility in biological media, which can affect nanomaterial mobility within the lung and hence its potential for systemic uptake. Taking these factors into account, RAC adopted the position of ECETOC (2006) and OECD SIDS (2004) of accepting SAS-DDS and SAS-PDMS as source substances to read-across to silanamine for human-health effects. Regarding the particle-size distribution of silanamine, the primary particles have the potential to aggregate to form strongly-bound or fused particles. Under conditions of normal handling and use, aggregates are the smallest stable particles. These aggregates can form agglomerates.

Industry commenters raised several scientific issues, including the particle size distribution of the SAS used in the inhalation studies and the nature of the effects following inhalation exposure. The toxicity of silanamine was low after both acute and repeated oral exposure. In the acute inhalation studies, the observed effects were restricted to the respiratory system and indicative of congestion and pneumonic oedema rather than systemic toxicity. It is therefore uncertain if the deaths after inhalation exposure represented a specific toxicity of silanamine or a non-specific, physical effect that resulted in suffocation. The OECD notes in its guidance 39 on inhalation toxicity testing that 'at very high concentrations, dry powder aerosols…tend to form conglomerates in the proximal nose causing physical obstruction of the animals' airways (e.g., dust loading) and impaired respiration which may be misdiagnosed as a toxic effect' (OECD, 2018). The Agency also notes that the ASASP (Association of Synthetic Amorphous Silica Producers) has commissioned a mechanistic study to address the concerns for


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inhalation toxicity. The study aims to obtain results under comparable conditions; and to achieve an understanding of the mechanism leading to the observed effects on animals. The study will include a detailed physico-chemical characterization of the test substance, a comprehensive characterization of the test atmospheres; and a (histo)pathological examination of the respiratory tract. The full results are expected to be available by the end of 2021. The Agency agrees with RAC’s assessment of the current data but notes the various uncertainties. Silanamine meets the criteria for classification for acute oral toxicity 2; H330 (Fatal if inhaled).

Specific target organ toxicity – single exposure (STOT SE)

Classification agreed by RAC: Not evaluated in the CLH report. During the public consultation, one Member State Competent Authority (MSCA) submitted a comment, noting that evidence from acute toxicity studies could be compared with CLP STOT SE criteria. RAC considered acute oral and inhalation toxicity studies as relevant for consideration of the classification for silanamine under STOT SE. RAC noted clinical signs of anorexia, eyes half-closed, chromodacryorrhea and blepharospasm (210 mg/m3), slight dyspnoea (2000 mg/kg bw) and irregular breathing. These signs were observed in both oral and inhalation studies, indicating stress caused by an unwell condition linked to exposure, but were not specific for any particular pathology. The most common findings during necropsy were discolouration of the lungs at 210 mg/m3 and 900 mg/m3, which was indicative of congestion, and pulmonary haemorrhage; and at 540 mg/m3 lungs were full of foam caused by the presence of particulates in lungs, indicating pneumonic oedema. Clinical signs persisted for four days before gradually reversing and although histopathology was not investigated, all clinical signs were connected with lung dysfunction. Concentrations with observed effects were close to the LC50 for acute inhalation toxicity. None of the findings met the criteria for classification in Category 1, 2 or 3; therefore, RAC concluded that classification for Specific Target Organ Toxicity – Single Exposure (STOT SE) is not warranted. Classification proposed by the Agency: The Agency agrees with RAC’s assessment of the data. Silanamine does not meet the criteria for classification for Specific Target Organ Toxicity – Single Exposure (STOT SE).


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Skin corrosion/irritation

Classification agreed by RAC: The DS referred to one in vivo skin irritation study (GLP-compliant, OECD TG 404) in rabbits in the CLH dossier. Scores for erythema and oedema were 0 when applied to intact and abraded skin. RAC also considered three literature reviews of information on SAS-HMDS, SAS-DDS and SAS-PDMS, where the maximum primary irritation index was between 0.25 – 0.44 out of 8 and all symptoms were reversible. With all skin irritation scores <2.3 (the minimum score for classification), RAC agreed with the DS that silanamine does not meet the criteria and therefore classification for skin corrosion / irritation is not warranted. Additional labelling The DS proposed to label silanamine with the phrase EUH066. This was based on a generally accepted but non-proven mode of action of silanamine, whereby hydrophobic SAS acts similarly to abrasive dusts by mechanically grinding the insects’ wax layer lipids. The cuticle then becomes enriched by the silica dust during treatment, thus reducing the wax layer of the cuticle. The hydrophobic nature of the silica intensifies adsorption to the insect's surface. As the human skin surface contains a layer of lipids of sebaceous and keratinocyte origin, the DS concluded the mode of action for silanamine as a biocidal active substance would also be relevant to humans. RAC also noted that repeated exposure to precipitated SAS without personal protective equipment can lead to mechanical irritation of the eye and drying/cracking of the skin. RAC therefore agreed with the DS that additional labelling with EUH066 (Repeated exposure may cause skin dryness or cracking) was warranted. Classification proposed by the Agency: The Agency agrees with RAC’s assessment on the data. Silanamine does not meet the criteria for classification for skin corrosion / irritation. The additional label EUH066 (Repeated exposure may cause skin dryness or cracking) should be applied.

Serious eye damage/irritation

Classification agreed by RAC: The DS referred to one eye irritation study (GLP-compliant, OECD 405) in rabbits with silanamine in the CLH dossier. The examination times were between 1 hour and 72 hours, with an irritation score of 0.25 when treated eyes were not rinsed and a score


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of 0.08 after rinsing. All signs of irritation were completely reversed within 24 hours. RAC also referred to an acute inhalation study where a single dose resulted in reversible chromodacryorrhea and blepharospasm. A further three literature reviews referred to information on the three hydrophobic SAS substances, where application resulted in no or slight but reversible irritation and had no evidence of irritation after washing the eyes. As all mean scores were <2 (minimum score for classification), RAC agreed with the DS that silanamine did not meet the criteria for serious eye damage / irritation and therefore classification was not warranted. Classification proposed by the Agency: The Agency agrees with RAC’s assessment of the data. Silanamine does not meet the criteria for classification for serious eye damage/irritation.

Respiratory sensitisation

Not assessed in the CLH report or RAC opinion.

Skin sensitisation

Classification agreed by RAC: The DS referred to a GLP- and OECD TG 406-compliant guinea pig maximisation test (maurer optimisation test) in the CLH dossier. Induction doses of 0.1% dilution were used on animals over three weeks (with Freund's adjuvant in week two and three) before a first challenge dose of 0.1% dilution after 13 days and a second challenge dose of 30% silanamine in Vaseline after a further 13 days were applied. No mortality or clinical signs were observed 48 hours after the final challenge. RAC recognised that the lack of reports of sensitisation in humans through decades of manufacture and use provided further reassurance of the substance's low skin sensitisation potential. Furthermore, SAS-DDS up to 30% as a pure substance and up to 7% in cosmetic products produced negative sensitisation results in several human repeat insult patch tests. As there was no evidence of hypersensitivity in animals or humans, RAC agreed with the DS that silanamine did not meet the criteria for skin sensitisation and, therefore, classification was not warranted.

Classification proposed by the Agency: The Agency agrees with RAC’s assessment of the data. Silanamine does not meet the criteria for classification for skin sensitisation.


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Specific target organ toxicity – repeated exposure (STOT RE)

Classification agreed by RAC: The DS referred to three oral feeding, two inhalation studies testing SAS-DDS in rats and one in vivo genotoxicity and gene mutation assay in rats with silanamine in the CLH dossier. No studies were completed using dermal administration and RAC concluded that as silanamine was not classified for skin irritation or skin sensitisation, toxicity was not likely after dermal exposure. The first 5-week (8-week for the high dose group) oral study (500, 1000, 2000 mg/kg bw/d) identified the liver as the target organ as there was occasional atrophy of liver epithelium, a loss of basophilic structure and a diminution of glycogen content in hepatocytes at mid to high doses. A second 6-month oral study using only one dose-level (500 mg/kg bw/d) revealed no treatment-related findings except a reversible stress reaction in the adrenals of treated rats which RAC concluded was of no toxicological significance. The final oral study was completed over one year, again with only one dose (100 mg/kg bw/d) and no treatment-related. RAC also noted a number of deficiencies in each study, such as: no tests complied with GLP or OECD guidelines; control groups were not used in all studies; statistical tests were not used; several tests did not give individual data; a low number of animals were used in one study. The 13-week (52-week recovery) inhalation study with SAS-DDS (GLP-compliant, OECD 413-like) identified the lungs as the target organ with an irreversible increase in septal cellularity and the following reversible findings at concentrations of 35 mg/m3: increased lung weight; swollen and spotted lungs; accumulation of granular material, cellular debris and leucocyte infiltration; granuloma-like lesions; increased collagen in the lung; signs of focal interstitial fibrosis (re-evaluation concluded this was not present); alveolar bronchiolisation; silicon detected in the lungs in a concentration-related manner. Clinical signs of nasal inflammation such as focal necrosis, rhinitis, slight degeneration of the olfactory epithelium and nasal irritation were also observed. A 14-day preliminary inhalation study that did not comply with any guidelines or GLP was used to identify the dosage for the 13-week study. The lungs were also identified as a target organ for the short-term investigation, with findings of respiratory distress at all concentrations (31, 87 and 209 mg/m3) after re-analysis, dyspnoea (87 mg/m3) and histological changes in the lungs related to alveolar inflammation. The DS also commented that the genotoxicity and gene mutation assay, with single intratracheal injection of up to 1.2 mg dust/lung increased the level of 8-OH-guanine in the lungs, leading to the assumption that chronic exposure may saturate the DNA repair mechanism. RAC also considered several literature studies alongside the inhalation studies referred to in the CLH dossier. Irregular breathing was a consistent clinical sign in all literature studies, as well as transient inflammation from histopathology analysis (in the alveolar region, local injury of the lungs, mediastinal lymph nodes and the nose), which RAC concluded was the expected adaptive response to inhalation of insoluble particles. After re-evaluation of the 13-week inhalation study it was concluded that


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interstitial fibrosis was not detected, correcting the main findings to fibrogenesis and extensive inflammation (Weber et al, 2018); however, other clinical signs such as increased collagen in the lungs, septal cellularity and alveolar bronchiolisation were not disputed. There was also increased activity of lactate dehydrogenase and N-acetyl-beta-D-glucosaminidase, which further supported the evidence that hydrophobic SAS resulted in tissue injury when inhaled. Irreversible interstitial fibrosis was observed in a 1-year study in monkeys using silanamine (Dow Corning, 1972; reviewed in Becker et al, 2013 and ECETOC, 2006) and irreversible local fibrosis was detected in an 8- or 12-month study in female rats using SAS-DDS at 50 mg/m3 (Degussa, 1962; reviewed in Becker et al, 2013). RAC recognised that both studies had several limitations such as lack of individual data and information on the number of animals. Alternatively, in the final examined literature review (Wacker, 1998; reviewed in ECETOC, 2006), there was no increase in birefringence, which is a common sign for interstitial fibrosis. RAC noted that silica levels decreased in the lungs and increased in the lymph nodes during recovery compared with immediately after exposure, leading to the conclusion that SAS was most likely solubilised and cleared to the lymph nodes where there was the adverse effect of histiocytosis in lung-draining mediastinal lymph nodes (reversible after 13-weeks recovery). A RAC minority opinion was published in addition to the RAC opinion, with the conclusion that classification under STOT RE 1 (H372) would be the most appropriate. The 13-week inhalation study was only tested using one dose level (35 mg/m3) with certain clinical signs showing slow, or no, recovery that recovery period for the original study is much longer (up to 52-weeks) than the guideline 4-week recommendation. It was noted that the re-evaluation also had many limitations such as: it only analysed certain animals in specific areas of the lung; the 30-year old cover slides were de-coverslipped which potentially damaged the original samples before being re-stained with hematoxylin and eosin (H&E) and re-coverslipped; H&E staining has lower sensitivity than Van Gieson staining in showing increases in fibre tissue; the recovery. Both inhalation studies showing interstitial fibrosis were not highly reliable but support the evidence that fibrosis could be a potential effect after exposure to chronic levels of SAS. Only one study (Wacker, 1998) tested exposure below the extrapolated guidance value for STOT RE 1 and resulted in severe lung effects such as histiocytosis in lung draining mediastinal lymph nodes from 10 mg/m3. As the study has a good reliability rating and the effects were only reversible after a long recovery period, some of RAC concluded that classification for STOT RE 1 (H372) was warranted). During public consultation, two industry associations, two individuals and two MSCAs submitted comments, with one MSCA agreeing with classification proposed in the CLH dossier and the remaining commenters opposing it. The MSCA in agreement with the CLH dossier indicated that the negative epidemiology study could not be used as evidence of no effect so adverse pulmonary effects in rats could not be ruled out, however, the second MSCA concluded that STOT RE 1 classification could not be excluded as the majority of studies did not expose animals to suitably low doses. The remaining two industry associations and two individuals were all


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against classification of silanamine under STOT RE. The first industry association concluded that effects could have been related to pulmonary overload and thus would not be considered intrinsic, but caused by ‘poorly soluble low toxicity particles.’ Furthermore, as no dose-response relationship was established and the majority of effects signifying inflammation were reversible, the requirements for classification were not met. Similarly, the second industry association felt the reversibility of the majority of effects meant that they should not be classed as adverse, in addition to crucial information being missed out of the CLH dossier, such as the re-evaluation of the 13-week study. Both individuals also commented on the missing re-evaluation from the CLH dossier, with the first also mentioning a number of occupational epidemiology studies that did not provide any indication of adverse lung effects. Additionally, the second individual concluded that the clearance of SAS particles from the lungs suggested that soluble particles had been used that, therefore, they would not cause the physio-pathological lung-overload phenomenon, which is known to cause persistent lung epithelial cell proliferation. In accordance with the CLP criteria, classification in Category 2 is permitted when evidence of toxicity with relevance to human health following repeated exposure in reliable animal studies is shown. As this was observed from the inhalation route, RAC agreed with the DS that silanamine met this criterion and that classification for Specific Target Organ Toxicity – Repeated Exposure (STOT RE) category 2 (H373; May cause damage to organs through prolonged or repeated exposure) was warranted. Classification proposed by the Agency: The Agency notes that there are some uncertainties in the assessment of this data. These include the apparent absence of systemic toxicity, with only adverse effects in the respiratory tract being observed, the absence of lung fibrosis and the possibility that the inflammation was a non-specific particle effect rather than an intrinsic property of the tested material. The Agency also notes that an investigation of the effects of silanamine after inhalation exposure is underway. The Agency agrees with RAC’s assessment of the current data but notes the uncertainties. Silanamine meets the criteria for classification for Specific Target Organ Toxicity – Repeated Exposure (STOT RE) 2 (lung; inhalation); H373 (May cause damage to organs through prolonged or repeated exposure).

Germ cell mutagenicity

Classification agreed by RAC: The DS referred to three in vitro and one in vivo studies in the CLH dossier. RAC concluded that SAS-HMDS (Aerosil R 812) was negative in an Ames test (non-GLP, OECD 471-like). RAC noted the limitations in the use of bacterial cells to


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investigate the mutagenicity of nanomaterials. A mammalian cell gene mutation test (GLP-compliant, OECD 476) with SAS-HMDS in mouse lymphoma L5178Y cells produced a positive linear trend in the presence of S9 mix but without a significant increase in mutation frequency. RAC also noted that the observed induced mutation frequency was lower than the global evaluation factor. The final in vitro study, a mammalian chromosome aberration test (non-GLP) in Chinese hamster ovaries with SAS-DDS also gave negative results. The in vivo genotoxicity and gene mutation assay in rats compared a single intra-tracheal injection of silanamine with a positive control of crystalline silica (quartz) dust (a known lung carcinogen). It evaluated four different parameters: measurement of DNA adducts (8-OH-guanine), markers of inflammation, histological analysis and presence of mutant p53 gene. Compared with a negative control, there was a significant increase in 8-OH-guanine during the first period of post-exposure; however, this had returned to the negative control level by day 90 while the positive control level remained significantly higher. The same trend was observed for acute inflammation, caused by an increased production of reactive oxygen species (ROS). After exposure to silanamine, it was also noted that there was no significant increase in accumulation of p53 while exposure to crystalline silica in the positive control resulted in significant accumulation of the gene over time. While all tested parameters were negative after exposure to silanamine, RAC could not rule out mutagenicity because of: inability to confirm that chronic exposure to a high level of silanamine would not lead to saturation of the DNA repair mechanism and cause mutations; lack of p53 detection did not ensure that no mutation has been induced; there was no in vivo test completed in somatic cells to complete the required testing for mutagenicity (CLP Regulation). During public consultation, two MSCAs submitted comments, both in support of the classification proposed in the CLH dossier. The first MSCA highlighted that the increase in 8-OH-guanine was evidence of a temporary change in DNA structure therefore, fulfilling the definition for genotoxicity, however agreed with RAC that this was not sufficient for classification. The second MSCA was in agreement that the data provided was not sufficient for classification but considered that a conclusion could not be drawn from the available data. As silanamine tested negative in all available in vitro and in vivo tests, RAC agreed with the DS that classification for germ cell mutagenicity was not warranted.

Classification proposed by the Agency: The Agency agrees with RAC’s assessment of the data. Silanamine does not meet the criteria for classification for germ cell mutagenicity.


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Carcinogenicity

Classification agreed by RAC: The DS referred to two studies in the CLH dossier. A two-year oral feeding carcinogenicity study in rats in which SAS-DDS was administered in the diet at one dose (100 mg/kg bw/d) (prior to OECD guidelines and not GLP) provided no evidence of neoplastic or non-neoplastic adverse effects. An epidemiological human health survey reported on effects after exposure to mainly hydrophilic SAS of unknown concentration and exposure time and was therefore outside the scope of the evaluation. No inhalation carcinogenicity study was available. During public consultation, two MSCAs submitted comments, both of the opinion that no classification can be proposed based on the absence of sufficient data. One MSCA also notes that inhalation carcinogenicity is a concern considering the increase in 8-OH-guanine DNA adducts in the lung. As there was no evidence that silanamine was carcinogenic in the available information, RAC concluded that classification for carcinogenicity was not warranted. Classification proposed by the Agency: The Agency agrees with RAC’s assessment of the data. Silanamine does not meet the criteria for classification for carcinogenicity.

Reproductive toxicity

Classification agreed by RAC: Adverse Effects on Sexual Function and Fertility The DS referred to a one-generation reproduction screening study in which silanamine was administered in the diet in rats. No impairment of reproductive performance, adverse effects on reproductive tissue or damage to foetal development were observed throughout the duration of the study. Some of the main deficiencies in the study noted by RAC were: only specific reproduction parameters were considered so current guideline requirements were not fulfilled; the mating period was too long to provide reliable data on male mating performance; only two males were used with a mating ratio of 1:5 instead of 1:2. As no positive results were observed in the available study for silanamine, RAC concluded that classification for adverse effects on sexual function and fertility was not warranted.


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Adverse Effects on Development No studies on silanamine or any of the hydrophobic read-across SAS approved by RAC were available. For the sake of completeness, RAC evaluated four oral gavage teratogenicity studies on hydrophilic silica gel (syloid, CAS number 112926-00-8) in mice, rats, hamsters and rabbits that were not in compliance with GLP or any current OECD guidelines. RAC concluded that these data indicated that hydrophilic SAS were not developmental toxicants. RAC concluded that classification for adverse effects on development was not warranted. Adverse Effects on or via Lactation Not assessed in the CLH report. RAC considered evidence from the one-generation reproduction screening study using SAS-DDS where offspring were examined post-partum and weekly during lactation for lesions indicative of teratogenicity, development and body weight effects. No differences were seen between exposed and unexposed groups of offspring in these metrics, nor in nursing behaviour, pup viability or pup growth to weaning. RAC concluded that classification for adverse effects on or via lactation was not warranted. Classification proposed by the Agency: The Agency agrees with RAC’s assessment of the data. Silanamine does not meet the criteria for classification for reproductive toxicity (effects on sexual function and fertility; effects on development; effects on or via lactation).

Aspiration hazard


Environmental hazards

Hazardous to the aquatic environment

Classification agreed by RAC: Rapid degradability of in-organic substances: RAC noted that silanamine was a highly hydrophobic and very stable substance which was insoluble in water and not accessible to biological transformation. The


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organic coating could make these substances susceptible to some form of biotic and abiotic degradation when compared with the non-surface-treated forms, however there were no data to support that hypothesis. The organic moiety formed only a small part of the substance (carbon content <5%) and degradation was only possible by physical means (for example, combustion would result in >99.5% silicon dioxide with only small amounts of water and carbon dioxide). RAC therefore concluded that no significant abiotic or biotic degradation of silanamine was expected as its chemical structure and composition was primarily of an inorganic rather than of organic nature. RAC also noted that no data were available on the potential ‘rapid removal’ of silanamine from the water column, a test which RAC considered to be more relevant than rapid degradability for these types of substances (metals and inorganic materials). Bioaccumulation: RAC agreed with analysis by the DS (ECHA, 2018 and in RCOM, ECHA 2019) regarding the low water solubility, low bioavailability via the water phase and low lipophilicity of silanamine, leading to the conclusion that it was not expected to be bioaccumulative. Following comments during public consultation that the bioavailability and uptake of such surface-modified nanoparticles was not well characterised or understood, RAC agreed that there was no actual data to unequivocally support such a conclusion since the methodologies for bioaccumulation testing of nanomaterials had not been finalized. However RAC still concluded overall, that silanamine was not expected to bioaccumulate significantly based on its chemistry and ‘biogeochemical cycle in nature’. Aquatic Toxicity: Acute aquatic toxicity studies on some apparently analogous synthetic amorphous silica (SAS) substances were summarised and tabulated in the RAC Opinion (ECHA, 2019). These studies covered acute toxicity to all of the main trophic groups of fish, aquatic invertebrates and algae:

• The static study on fish (Danio rerio, cited as former name Brachydanio rerio) was conducted according to OECD TG 203 using the substance SAS-DDS (‘Aerosil R974’). The nominal test concentrations were a control, 1000 and 10000 mg/L. Test suspensions were stirred in test vessels for about 20 hours on a magnetic stirrer at 25°C and then allowed to stand for 4 hours. It was apparent to RAC that the ecotoxicity concentrations were loading rates rather than actual concentrations and that no analytical measurement occurred of dissolved or suspended material, settling, aggregation, agglomeration or any other transformation processes. RAC noted a number of other deviations from the protocol and the OECD Guidance document (No. 23) on aquatic toxicity testing of difficult substances and mixtures (OECD, 2002). This included that a 48-hour stirring period was recommended to achieve the maximum dissolved concentration and non-dissolved substance should be separated and removed


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before testing (which was not done in this case). This may however have not been appropriate if the aim was to test the suspended nanomaterial. Nevertheless, RAC felt that the substantial nominal overdoses were likely to have achieved sufficient maximum dissolved concentrations. Also other parameters (control mortality, oxygen saturation, pH fish size and weight) were all in accordance with test validity criteria. Therefore, RAC agreed that the study gave a reasonable reflection of the lack of toxic effects in fish. No mortalities and no sub-lethal effects occurred at any of the nominal concentrations tested, and the test substance was presumed not to be acutely toxic to Brachydanio rerio within the exposure conditions of this test. The nominal 96-hour LC50 was > 10000 mg/L.

• Similarly, a 24-hour static acute test was conducted on Daphnia magna using SAS-DDS (‘Aerosil R974’) according to OECD TG 202. The nominal test concentrations were a control, 1000 and 10000 mg/L and again there were no analytical measurements conducted. Test suspensions were stirred in test vessels for about 20 hours. All of the concentrations were tested un-filtered but the 10000 mg/L concentration was also tested after a filtration. No effects were seen on immobility and no abnormal behaviour was noted in the test organisms at any dose. RAC therefore concluded that the test substance was not acutely toxic to Daphnia magna and the nominal 24-hour EC50 was > 10000 mg/L under these test conditions.

• In the test on algae, Desmodesmus subspicatus (cited as former name Scenedesmus subspicatus) was exposed for 72 hours under static conditions according to OECD TG 201. The nominal test concentrations were a control, 100, 1000 and 10000 mg/L and there were no analytical measurements of the test material conducted. Test suspensions were incubated in a shaking machine for 24 hours and then all of the concentrations were filtered. Cell concentration in controls increased at least by a factor of 16 within 3 days so meeting this validity criterion. In the treated groups, no reduction in growth rate was observed after 72 hours. RAC noted deficiencies regarding the absence of any explanation for a pH deviation (about 3 units) and the absence of cell concentration results for each flask at each measuring point, along with the variation coefficient for replicates of controls and test concentration. However, these were considered of limited importance for the outcome of the study. Therefore, the test substance was presumed to not be acutely toxic to algae and not to inhibit the growth of the Desmodesmus subspicatus within its aqueous solubility, as tested. The nominal 72-hour ErC50 was > 10000 mg/L.

RAC recognised there were several significant deficiencies with the studies regarding the evaluation of environmental hazards. There were no studies on silanamine itself,


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only with read across substances which had a slightly different surface coating. However, the read-across justification was supported by RAC as explained in their Opinion (ECHA, 2019). Whilst the more recently developed OECD protocols and guidance for nanomaterial testing were not followed or discussed in the CLH dossier, RAC concluded overall that it was ‘rather unlikely’ that silanamine would cause an acute hazard to aquatic organisms. Consequently, RAC proposed no classification for aquatic acute hazard due to insufficient evidence of likely harmful acute effects according to CLP criteria. RAC noted that no chronic aquatic toxicity studies were available for silanamine, or other hydrophobic SAS, for any of the three trophic levels. RAC observed that no effect was seen in the acute ecotoxicity tests performed at maximum nominal concentrations of 10000 mg/L and based on a weight of evidence approach, silanamine had a low potential to bioaccumulate. Moreover, the conventional biodegradation studies designed to test organic substances were not considered applicable for such inorganic substances considering their high stability and inertness. Amorphous silica was not considered rapidly degradable in general although the surface treated silanamine could exhibit some degradation of its methylated coating. Overall however, based on a weight of evidence, RAC proposed no classification for aquatic chronic hazard due to insufficient evidence of likely harmful chronic effects according to CLP criteria. RAC Opinion: Based on limited information on the environmental fate and acute toxicity of silanamine or similar substances, RAC agreed that the substance did not require any Acute or Chronic aquatic hazard classification. RAC responded to comments made during public consultation questioning whether, in the absence of information and appropriate nanomaterial testing of silanamine, a Chronic 4 ‘safety net’ classification was appropriate. RAC noted the mode of action was based on the physical and functional impairment or destruction of the lipid-wax layer cuticle, rendering the target organism unprotected from water loss. Although there could be some effects on both aquatic and terrestrial organisms following chronic exposure, RAC did not believe this was supported by the available acute aquatic toxicity test results. Whilst silanamine was a poorly soluble substance which was not rapidly degradable in normal terms, RAC also did not consider it had a significant potential to bioaccumulate. Overall therefore, in a weight of evidence approach, RAC considered that an Aquatic Chronic 4 classification was not warranted. Classification proposed by the Agency: The Agency agrees that as silanamine is mostly (≈ 95%) an inorganic substance and so the standard degradation testing and CLP criteria for rapid degradability of organic substances are not applicable. Whilst some degradation of the methylated


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surface coating may occur, this is not considered to sufficiently affect its likely fate or effects on aquatic organisms (judged by the available acute toxicity data on un- coated analogues) to influence its aquatic hazard. No transformation/dissolution testing or dispersion stability testing according to OECD GD 29 (2001), TG 318 (2017) or GD 318 (2020 or earlier drafts) had been conducted and RAC noted that no case has been made regarding ‘rapid removal’ of silanamine from the water column, as proposed for metals and other inorganics. However, recent discussions with ECHA and industry and other stakeholders, have concluded that there is currently no clear case for using rapid removal of inorganic material as a surrogate for rapid degradation of organic substances. This has therefore not been considered further by the Agency. In terms of bioaccumulation potential, the Agency notes that this has not been fully considered according to the latest developing ECHA guidance on the bioaccumulation testing and assessment of nanomaterials, however the dossier was compiled before this was available. Silanamine is practically insoluble in water (< 1 µg/L) and octanol and therefore should have a low bioavailability via both the water and lipid phase. It is essentially amorphous silica formed from polymerically bound silicon dioxide which is abundant in nature. No experimental data are available to derive a bioconcentration factor (BCF) in aquatic species and no log Kow is available on silanamine. However, the standard CLP log Kow trigger of ≥4 for organic lipophilic substances is not considered applicable for inorganic molecules. There are some concerns that nanomaterials may accumulate in organisms via different pathways to traditional lipid bioavailability mechanisms and also whether the organic hydrophobic coating of silanamine could affect its uptake in aquatic organisms. However, the Agency agrees overall that based on the existing weight of evidence, silanamine is considered to have a low bioaccumulation potential for hazard classification purposes. The Agency notes that acute studies on fish, aquatic invertebrates and algae are available and that these indicate low acute toxicity with nominal LC/EC50s all > 10000 mg/L. A number of deviations from the standard test guidelines were noted by RAC but they considered these would not significantly affect the study interpretations. The Agency notes that, in any case, the standard test guidelines and criteria, including for difficult to test substances, may not be so applicable for nanomaterials where it might actually be intended to test a suspension. However, the studies were conducted before more recent OECD and ECHA guidance on ecotoxicity testing and assessment of nanomaterials (e.g. OECD GD 317, 2020) was available. The substances tested were also not identical to the hydrophobically coated silanamine, however the DS and RAC have made a case in the RCOM and Opinion (ECHA 2019) for read-across of these data, which, on balance, the Agency can agree with. Additionally, silanamine is complicated to analyse with an LOD in water (1 mg/L) being much higher than the solubility limit (< 1 µg/L). RAC considers that the solubility limit under these test conditions was likely achieved due to an excess of substance in the nominal concentrations. L/EC50s were expressed as nominal


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concentrations and not set to the solubility limit because the substance is practically insoluble in water and effects on aquatic organisms are not observed at higher concentrations. Thus, endpoints based on the solubility limit were considered not to reflect the actual environmental effect. Overall the Agency can agree with RAC that, on balance, the available acute data are a fair reflection of the likely low acute toxicity of silanamine to aquatic life. As all nominal LC/EC50s are > 10000 mg/L, the Agency agrees that no classification for Acute Aquatic hazard is required for silanamine. The Agency notes that there are no chronic ecotoxicity data available on silanamine or similar substances. Whilst the substance does not degrade in the same sense as with an organic substance, and so may persist in aquatic environments, it is also not expected to bioaccumulate and no significant effects were seen in the acute tests on fish, invertebrates or algae at up to a nominal 10000 mg/L. The Agency agrees with RAC therefore, that on balance, no classification for Chronic Aquatic hazard is required for silanamine. This lack of any proposed aquatic classification includes the lack of a Chronic 4 ‘safety net’ classification as the CLP criteria for this are not strictly met. However, whilst there are no other indications of likely adverse effects, there is still uncertainty in the relevance of the available data. It is possible that silanamine, or similar nanoforms of synthetic amorphous silica may be required to have updated dossiers or be re-registered under biocides or industrial chemicals legislation (REACH) using more up to date test guidelines and guidance specifically relevant to nanomaterials. Should any such further data become available, then the aquatic hazard classification of silanamine may need to be reconsidered.

Other hazards

Hazardous to the ozone layer



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Overall conclusion

The Agency has evaluated the RAC Opinion, its rationale and any additional scientific evidence that may have been made available to HSE against the criteria for classification and labelling in the GB CLP Regulation and technical guidance. The Agency technical report agrees with the classification proposed by RAC for the following hazards: Acute Tox 2; H330 (Fatal if inhaled) with an ATE of 0.45 mg/L (dusts or mists) STOT RE 2 (lung; inhalation); H373 (May cause damage to lungs through prolonged or repeated exposure via inhalation) NOT CLASSIFIED for physical hazards, acute toxicity (via the oral and dermal routes of exposure), STOT SE, skin corrosion/irritation, serious eye damage/irritation, skin sensitisation, germ cell mutagenicity, carcinogenicity, reproductive toxicity and hazardous to the aquatic environment. The additional label EUH066 (Repeated exposure may cause skin dryness or cracking) should be applied. The Agency technical report disagrees with the classification proposed by RAC for the following hazards: Not applicable

Overall, the conclusion is to agree with the RAC opinion.


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References

OECD (2018). Guidance document on inhalation toxicity studies. Series on Testing and Assessment No. 39. ENV/JM/MONO(2009)28/REV1 OECD (2002), Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD Series on Testing and Assessment, No. 23, OECD Publishing, Paris ECHA (2017) Guidance on the application of the CLP criteria. Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, version 5.0, ref: ECHA-17-G-21-EN. Available at https://www.echa.europa.eu/ For all other references, please see the EU CLH report and the EU RAC opinion (available at: https://echa.europa.eu/registry-of-clh-intentions-until-outcome) ECHA (2018) CLH report (including Annexes): Proposal for Harmonised Classification and Labelling Based on Regulation (EC) No 1272/2008 (CLP Regulation), Annex VI, Part 2. Substance Name: Silanamine; Date:2018; Accessed date: 25/2021 ECHA (2019) Committee for Risk Assessment (RAC) opinion (including Annexes) proposing harmonised classification and labelling at EU level of Silanamine; Reference CLH-O-0000006735-67-01/F; Date: 05/12/2019 Accessed date: 25/2021 Documents published as part of the EU CLH process: Source: European Chemicals Agency, http://echa.europa.eu/

https://echa.europa.eu/registry-of-clh-intentions-until-outcome


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Glossary of terms used in Agency technical reports

Agency, the HSE, acting in its capacity as the GB CLP Agency AR Applied radiation ATE Acute toxicity estimate BCF Bioconcentration factor BOD Biological Oxygen Demand bw Body weight CAR Competent Authority Report CAS Chemical Abstracts Service CI Confidence interval CL Confidence limits

CLH Harmonised Classification and Labelling CLP Classification, labelling and packaging (of substances and mixtures) CO2 Carbon dioxide COD Chemical Oxygen Demand CV Coefficient of Variation d Day DAR Draft Assessment Report DDS Dimethyldichlorosilane DOC Dissolved Organic Carbon DS Dossier Submitter DT Dissipation time OR degradation time (also DissT or DegT where

apparent) DT50 Dissipation half-life OR degradation half-life (hours or days), see

also above dw Dry weight ECHA European Chemicals Agency ECx x% effect concentration EFSA European Food Safety Authority ErCx x% effect concentration based on growth rate EU European Union GLP Good Laboratory Practice h Hours HDMS Hexamethyldisalazane H&E Hematoxylin and Eosin KOC Organic carbon-water partition coefficient

KOW Octanol-water partition coefficient LCx x% lethal effect concentration MCL Mandatory Classification and Labelling M-factor Multiplying factor MSCA Member State Competent Authority MW Molecular weight NOEC No-observed effect concentration


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OECD Organisation for Economic Co-operation and Development PDMS Polydimethylsiloxane QSAR Quantitative structure-activity relationship RAC Risk Assessment Committee RAR Renewal Assessment Report RCOM Response to comments document REACH Registration, Evaluation, Authorisation and Restriction of

Chemicals regulation ROS Reactive Oxygen Species SAS Synthetic Amorphous Silicas STOT-RE Specific target organ toxicity – repeated exposure STOT-SE Specific target organ toxicity – single exposure

TG Test Guideline US EPA United States Environmental Protection Agency wt Weight

wwt Wet weight