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1
Evaluation of Alternative Methods for Assessing Acute Toxicity of Mixtures
Raja S. Settivari, BVSc&AH, PhD, DABT
Dow Chemical Company
2
Purpose
Evaluation of alternative methods for:
Testing formulations/mixtures for acute contact toxicity endpoints
Application of alternative methods for sustainable formulations development
3
Development of Alternative Testing Methods
An active area of research to develop and validate alternative methods
Acute toxicity and Receptor binding endpoints
Increased focus to: Explore applicability domain and identify limitations/gaps
Identify complementary assays and develop integrated testing strategies (ITS)
Performance of an ITS depends on the extent to which the different assays are able to compensate for each other’s technical limitations
Promote utilization and acceptance globally
Application of alternative methods are growing
Endocrine Screening program
Dermal sensitization, Dermal irritation, Eye irritation
Lautenberg Chemical Safety Act: Non-animal models
4
Testing strategies
Tools: QSAR, Analog ID, Read Across, Data mining
(Internal and publically available data)
Tier 1 -Cheminformatics
In vitro predictive assays
(selected based on specific question/need)
Tier 2 –In vitro biological profiling
Knowledge
integration
In vivo (animal) assays
(selection based on regulatory need)
Tier 3 – Standard Regulatory Toxicology
Imp
lem
en
tati
on
Approach that supports implementation of IATAs
5
ICCVAM. 1999. NIH Publication No. 99-4494 ICCVAM. 2010. NIH Publication No. 11-7709
Urbisch et al. 2015. Reg Tox Pharm 71:337-351. Dumont et al. 2016. Tox In Vitro 34: 220-228
ENV/JM/MONO(2013)12/PART2 Hoffmann et al. 2017 submitted
Hazard 72%-82%
Potency 54% - 60%
Hazard ~72%
Potency ~60%
GPMT / Buehler LLNA
Reproducibility of Multiple Tests (~100 chemicals)
Hazard ~78%
Potency ~62%
Accuracy Against Human Clinical Data (~150 chems)
Draize Eye Irritation Test Limited within- and between-laboratory reproducibility High variability for moderate to mild irritating compounds
6
A way to deliver an active ingredient (AI) in a usable form
Consists of multiple components Active Ingredient(s) (AI)
Inert ingredients Surfactants Dry fillers and carriers Solvents, Thickeners, Biocides, Odorants, Anti-freeze, Stabilizers, Dyes, Pigments
Effective implementation of alternative methods may lead to: Development of sustainable products
Greater reduction in animal usage
Formulation/Mixtures For Everyday Use
• Components with different toxicity potential • Compatibility issues at higher conc. • Can’t be defined by Molarity
Challenges
7
Dermal Sensitization The key mechanistic events underpinning the skin sensitization process that leads to
Allergic Contact Dermatitis (ACD) in humans have been identified
1
4
5 2
3
DPRA KeratinoSens
M&K
Buehler
LLNA
Human Cell Line
Activation Test
Myeloid U937 Skin Sensitization Test;
IL-8 luc Test
8
8 agrochemical AIs and 10 corresponding multi-component formulations (Commercial quality)
All samples had prior in vivo Buehler, M&K or LLNA results
Three different types of formulations were tested in the KeratinoSens and DPRA:
Soluble Concentrate (SL) AI is presented as a water soluble salt
Emulsion Concentrate (EC) AI is solubilized in an organic solvent to be applied as an emulsion after
dilution in water
Suspension Concentrate (SC) Low water soluble and high melting point AI is suspended in water
Dermal Sensitization
9
KeratinoSens Assay
Cells: HaCaT cells with stably integrated ARE-luciferase reporter gene
Dose Levels: Limit concentration of 2000 µM or 4 mg/mL or 0.4%
Provides concentration-response information (12 concentrations)
Study measurements/readouts (separate plates):
Luciferase induction
Cytotoxicity
Keap1
S S
Nrf2
Nrf2
ARETarget Genes
Luciferase
Activated state
Figure adapted from Natsch, 2010
Keap1
SH SH
Nrf2
Nrf2Ub
Non-activated state
HaCaT cell line
Stably integrated ARE reporter gene
Keap1
S S
Nrf2
Nrf2
ARETarget Genes
Luciferase
Activated state
Figure adapted from Natsch, 2010
Keap1
SH SH
Nrf2
Nrf2Ub
Non-activated state
HaCaT cell line
Stably integrated ARE reporter gene
Nucleus
Cytoplasm
Ub-dependent proteasomal
degradation
10
Approaches for Dose Selection
Approach 1: • Based on the MW and concentration of AI in the formulation
(ranged from 4.9% to 81.8%)
• Potential challenges: • Results in testing co-formulants at higher concentrations • Test material compatibility issues and false positive results
Approach 2: • Test at a constant maximum “mg/ml” concentration • Based on the total formulation (considered formulation as single entity) • Avoid testing the co-formulants at higher concentrations
• Potential challenges:
• False negative results
Results were compared to existing in vivo data and human data (when available)
11
Test Material Type AI conc, %
In vivo data
KeratinoSens assay data
(Corrected to MW and purity)
(Approach 1)
KeratinoSens assay data
(Constant max conc)
(Approach 2)
GF-700 (AI: Acetochlor) EC 81.8 Sensitizer Sensitizer Sensitizer
GF-1478 (AI: Meptyldinocap) EC 34.4 Sensitizer Non-sensitizer Sensitizer
GF-2870 (AI: Triclopyr choline) SL 54.6 Sensitizer Non-sensitizer Sensitizer
XRM-4714 (AI: Triclopyr butotyl) EC 61.8 Sensitizer Sensitizer Sensitizer
GF-871 (AI: Aminopyralid
triisopropanolammonium SL 40.1 Non-sensitizer Non-sensitizer Non-sensitizer
GF-2000 (AI: Clopyralid
monoethanolammonium) SL 43.2 Non-sensitizer Non-sensitizer Non-sensitizer
EF-1343* (AI: Florasulam) SC 4.91 Non-sensitizer Sensitizer Non-sensitizer
GF-837 (AI: Methoxyfenozide) SC 22.8 Non-sensitizer Non-sensitizer Non-sensitizer
GF-1243 (AI: Oxyfluorfen) EC 22.3 Equivocal Sensitizer Non-sensitizer
GF-1191 (AI: Oxyfluorfen) EC 22.6 Equivocal Sensitizer Non-sensitizer
Testing co-formulants at higher concentrations resulted in false predictions in Approach-1
Approach-2 provided better predictions compared to in vivo results
Settivari et al., RTP, 2015
KeratinoSens Test Results for Formulations
12
Formulations
with single AI KeratinoSens
analysis
n % Correct
predictions
Correct 27 96.4%
False Positives 0 NA
False Negatives 1 3.6%
Formulations
with multiple AIs KeratinoSens
analysis
n % Correct
predictions Correct 7 78% False Positives 1 11% False Negatives 1 11%
KeratinoSens Test Results for Formulations
In vivo classification
for respective AIs In vitro KeratinoSens results
In vivo
results
Formulations with single AI LLNA/Guinea pig studies EC1.5
(µM)
Cell
viability
(%)1 Interpretation In vivo studies
Acetochlor-EC Sensitizer 1.43 >70% Sensitizer Sensitizer
Meptyldinocap-EC Sensitizer 1.79 >70% Sensitizer Sensitizer
Triclopyr Butoxyethyl Ester-EC Sensitizer 71.25 >70% Sensitizer Sensitizer
Triclopyr choline-SL Sensitizer 623.09 >70% Sensitizer Sensitizer
Cyhalofop butyl-EC-1 Non-sensitizer 26.4 >70% Sensitizer Sensitizer
Cyhalofop butyl-EC-2 Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Aminopyralid
triisopropanolammonium-SL Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Clopyralid monoethanolammonium-SL Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Florasulam-SC Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Methoxyfenozide-SC Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Oxyfluorfen-EC-1 Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Oxyfluorfen-EC-2 Non-sensitizer 545.21 >70% Non-sensitizer Non-sensitizer
Propiconazole-SC Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Spinetoram-SC Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Spinosad-SC Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Sulfoxaflor-SC Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Formulations with multiple AIs
Triclopyr salt + Aminopyralid-SL Sensitizer + Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Triclopyr ester + Aminopyralid-EC Sensitizer + Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Triclopyr ester + Florasulam-EC Sensitizer + Non-sensitizer NA2 >70% Non-sensitizer Sensitizer
Aminopyralid + Halauxifen methyl-SC Non-sensitizer + Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Aminopyralid + Clopyralid-SL Non-sensitizer + Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Aminopyralid + Fluroxypyr-EW Non-sensitizer + Non-sensitizer 114.1 >70% Sensitizer Sensitizer
Picloram potassium + 2,4-D-SL Non-sensitizer + Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Aminopyralid + Picloram potassium +
Clopyralid-SL Non-sensitizer + Non-sensitizer
+ Non-sensitizer NA2 >70% Non-sensitizer Non-sensitizer
Aminopyralid + Picloram potassium +
Fluroxypyr-EW Non-sensitizer + Non-sensitizer
+ Non-sensitizer 140.3 >70% Sensitizer Non-sensitizer
13
Direct Peptide Reactivity Assay (DPRA)
In chemico procedure
Measures depletion of a target peptide following incubation with a test material.
Depletion quantified using HPLC-UV.
O
O
F
F
F
O
O
F
F
F
Hapten
E
Protein
O
O
F
F
F
Protein
Hapten O
O
F
F
F
:Nu
The hepta-peptides are incubated with test material at for 24 h.
Lysine Peptide (K): Ac-RFAAKAA-COOH (Peptide:TM – 1:50 ratio)
Cysteine Peptide (C): Ac-RFAACAA-COOH (Peptide:TM – 1:10 ratio)
Interpretation criteria:
Avg of Cysteine & Lysine model: Peptide depletion threshold – 6.38%
Cysteine model: Peptide depletion threshold – 13.89%
14
DPRA Test Results for Formulations
Formulations
with single AI DPRA analysis
n % Correct
predictions
Correct 14 87.5%
False Positives 0 NA
False Negatives 2 12.5%
Formulations
with multiple AIs DPRA analysis
n % Correct
predictions Correct 8 89% False Positives 0 NA False Negatives 1 11%
Liquid Chromatography with Tandem Mass Spectrometry (LC/MS-MS) to improve sensitivity
and specificity
Selected in vitro methods provided good predictions for testing formulations
15
Active
Ingredients (AI)
Formulations w/ single
AI
Formulations 2 or
more AIs
N 20 16 9
KeratinoSens 85% 100% 78%
DPRA 91% 87.5% 89%
GHS Threshold - 87.5% 78%
WoE (2 of 3) 100% 100% 89%
Accuracy relative to
in vivo data
Integrated Testing Results for Formulations
Threshold-based approach (Formulations)
GHS approach: Read-across from sensitizing ingredients present ≥ 1% to the mixture
A substance testing positive or negative in any two methods are classified as
sensitizers or non-sensitizers, respectively
Key learnings
In silico:
Closer attention to applicability domain
KeratinoSens:
Dose response, Cytotoxicity, LogKoW, Physical properties of the test substance
DPRA:
Solvent, Non-covalent interaction b/n TM and peptide
Conclusion
Selected alternative methods provided promising performance for evaluating skin sensitization potential of multi-component mixtures
17
Ocular Irritation Assays 1. Organotypic models
Hen’s egg test – Chorioallantoic membrane test
Isolated rabbit eye test
Isolated chicken eye test (OECD TG 438)
Bovine corneal opacity and permeability test (OECD TG 437)
2. Cell-based models
Short Time Exposure In Vitro Test Method (OECD TG 491)
Red blood cell hemolysis test
Cytosensor Microphysiometer
Fluorescence leakage test (OECD TG 460)
Neutral red release assay
3. Reconstructed human tissue models Reconstructed Human Cornea-like Epithelium Test (OECD TG 492)
18
Ocular Irritation study:
Selected 64 formulations for evaluating the performance of the NRR and EpiOcular assays
Selection based on availability of high quality in vivo data
11 types of formulations
Represented both solids and liquid type formulations
Settivari et al., RTP, 2016
19
Neutral Red Release (NRR) Assay Performance
Accuracy (%) Sensitivity (%) Specificity (%) Sample size
78 85 76 64
20
G H S
C a t . 1 + 2
G H S
N o t c la s s if ie d
0
2 0
4 0
6 0
G H S c a te g o r ie s ( in v iv o d a ta )
ET
40
va
lue
s
G H S C a t N C
G H S C a t 1 + 2
EpiOcular Assay Performance
Accuracy
(%)
Sensitivity
(%)
Specificity
(%)
Sample
size
65 58 75 51
21
Tiered Testing Approach
Accuracy
(%)
Sensitivity
(%)
Specificity
(%)
Sample
size
75 73 77 64
Tiered Testing Approach for Ocular Irritation
Key learnings
In silico: Closer attention to applicability domain
NRR assay:
Dose response, Physical properties, Coloring agents
EpiOcular:
Damaged tissue during shipping, Physical properties, Coloring agents
Conclusion
Selected alternative methods provided promising performance for evaluating ocular irritation potential of multi-component mixtures
Inclusion of methods to assess post-exposure recovery may improve predictive performance of alternative methods
23
OECD 435: In vitro membrane barrier test method OECD 431: Reconstructed human epidermis test method OECD 430: Transcutaneous electrical resistance test method OECD 439: Reconstructed human epidermis test method
Dermal Irritation Testing Strategy
Corrosives
OECD 435/431/430
Corrosive STOP
+ - Irritants
OECD 439
+
Irritant (Cat-2)
-
NC
Non irritant STOP
No testing for chemicals with extreme phys-chem properties
8/10 Formulations were correctly predicted compared to existing in vivo data Good predictions for corrosives, however lower predictions for moderate and
milder irritants
24
Applications of In Vitro Assays Evaluation of multiple molecules as part of lead-candidate prioritization
during early stage development
In silico data is used to determine metabolism requirement and selection of in vitro tests. *GHS Threshold approach as part of WoE assessment for mixtures
25
Applications of In Vitro Assays
Formulations development Co-formulants testing to identify least toxic alternatives
Determine potential threshold (non-toxic) levels for co-formulants
Hypothesis driven research to develop sustainable formulations
Selected alternative methods identified formulations with reduced sensitization potential
26
Conclusions
Selected testing methods demonstrated promising performance for evaluating acute toxicity of multi-component mixtures.
Establishing a sound scientific understanding on the applicability domain of the alternative test methods improves confidence and public trust.
Resource efficient and Reduce the requirement for animal testing. Applicable at earlier stages of sustainable formulation development.
27
Acknowledgements
Dow Chemical
Darrell Boverhof
Sue Marty
Sean Gehen
Marco Corvaro
Dan Wilson
Ricardo Acosta Amado
Nicolo Visconti
Fagen Zhang
Jeremy McFadden
Matt Koehler
Julie Wheatley
Givaudan
Andreas Natsch
28
Thank you