Isobutylbenzene

Endpoint summary

Description of key information

The skin sensitizing potential of the test substance was assessed using an in vitro OECD guideline testing strategy comprising the following assays:

- Direct Peptide Reactivity Assay (DPRA),

- Keratinocyte Activation Assay (LuSens) and

- Dendritic Cell Line Activation Assay (h-CLAT).

The DPRA and LuSens assay were conducted under GLP according to the respective OECD guideline.

The results were as follows:

- DPRA: negative/inconclusive (due to the limited solubility of the test substance with both peptides)

- LuSens: negative

- h-CLAT: waived, because due to the substance properties (test substance has a Log KOW > 3.5) it was not possible to recieve a conclusive result

In addition, a quantitative structure-activity relationship (QSAR) system for the estimation of the skin sensitization potency that incorporates skin metabolism and considers the potential of parent chemicals and/or their activated metabolites to react with skin proteins was used. Two QSAR programs (TIMES-SS and QSAR Toolbox) predicted the substance not to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan - Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Certificate produced by "Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht" Rheinland Pfalz

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE; batch No.: EXP-18/11/346
- Purity test date: 18/11/2015
- Log Kow: 3.94 (calculated); 4.8 at 23°C and pH 6.0 (measured)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as a 100 mM (considering a molecular weight of 134.22 g/mol and a purity/contents of 99.9%) preparation in acetonitrile within 4 hours of preparation of test-substance samples. After short stirring the test substance was soluble in the vehicle. All test-substance preparations and dilutions were made in glass vials.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in acetonitrile

Details on the study design:

TEST SYSTEM
- Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol); Lysine-(K-)containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K­ containing peptide). The peptide stock solution were used for preparing the calibration samples, the test-substance and control samples.
- Source: The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and/or RS Synthesis, Louisville KY, USA and/or JPT Peptide Technologies GmbH, Berlin, Germany) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

CONTROLS
- Vehicle control: acetonitrile: Set A) performance control (analyzed together with the calibration samples without incubation); Set B) Stability control (placed at the very start and ending of the sample list for HPLC analysis); Set C) for calculation of the peptide depletion (analyzed with the samples)
- Positive control: ethylene glycol dimethacrylate (EGDMA; CAS no. 97-90-5) (prepared as a 50 mM solution in acetonitrile)
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide

VEHICLE
- Vehicle: acetonitrile
- Reason for choice of the vehicle: The test substance was soluble in acetonitrile

SAMPLE PREPARATION
- Peptide stock solutions were mixed with the test substance or positive control or vehicle control at a ration of 1:10 (0.5 mM C-peptide, 5 mM test substance) or 1:50 (0.5 mM K-peptide, 25 mM test substance)

EXPERIMENTAL PROCEDURE
- No. of replicates: 3 (for each peptide)
- The test substance was prepared at a 100 mM concentration. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containg peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).
- Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis
- Samples were incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours
- The remaining non-depleted peptide concentration was determined by HPLC with gradient elution and UV-detection at 220 nm about 24 hours after sample preparation (for details on HPLC conditions see tab. 3). The analysis time itself did not exceed 30 hours.
- Calibration samples of known concentration (dissolved in 20% acetonitrile in the respective buffer), prepared from the respective peptide stock solution used for test-substance incubation were measured before analysis of the test-substance samples with the same analytical method (for details see tab. 1)

DATA EVALUATION (for detailed formulas see "Any other information on material and methods")
Calculation of the peptide concentrations:
- For each peptide a calibration curve is generated from the measured peak areas of the calibration samples of known peptide concentration. The peptide concentration of the samples is calculated with the respective calibration curve using linear regression (b = axis intercept; m = slope).
Calculation of the peptide depletion:
- The mean peptide depletion for each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance. When a negative value for C- or K-containing peptide depletion is obtained the value is considered zero for calculation of the mean peptide depletion. The mean peptide depletion of a test substance is calculated as the mean value of C-containing peptide depletion and K-containing peptide depletion.

ACCEPTANCE CRITERIA
- The standard calibration curve should have an r² >0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be < 15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
- The positive control should cause depletion of both peptides comparable to historic data.

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

due to the limited solubility of the test substance

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see tab. 9a+b

Table 6: Peptide depletion for C-peptide

Reaction with cysteine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean; SD
NC: ACN	2.35	-3.88	1.52	0.00; 3.38
Test substance	-3.92	-3.19	0.29	-2.27; 2.25
PC: EGDMA in ACN	63.85	67.91	71.14	67.63; 3.65

Table 7: Peptide depletion for K-peptide

Reaction with lysine-peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean; SD
NC: ACN	0.23	-0.49	0.27	0.00; 0.43
Test substance	-0.93	-0.66	0.40	-0.40; 0.70
PC: EGDMA in ACN	13.80	13.63	13.50	13.64; 0.15

Table 8: Mean peptide depletions

	Cysteine-Peptide   mean depletion [%] SD [%]		Lysine-Peptide   mean depletion [%] SD [%]		mean of both depletions [%]
Test substance	-2.27	2.25	-0.40	0.70	0.00
PC: EGDMA in ACN	67.63	3.65	13.64	0.15	40.64

Table 9a: Historic control data of vehicle control (acetonitrile) (not including present study)

	C-peptide concentration [mM]	K-peptide concentration [mM]
Min	0.451	0.486
Max	0.511	0.536
Mean	0.484	0.508
SD	0.015	0.011
n	42	40

Table 9b: Historic control data of positive control (EGDMA, 50 mM in acetonitrile) (not including present study)

	C-peptide concentration [mM]	C-peptide depletion [%]	K-peptide concentration [mM]	K-peptide depletion [%]
Min	0.144	43.32	0.395	6.79
Max	0.282	70.07	0.491	21.52
Mean	0.216	55.20	0.445	12.90
SD	0.032	6.23	0.020	3.10
n	32		30

Interpretation of results:

study cannot be used for classification

Remarks:

limited solubility of the test substance leading to emulsive samples

Conclusions:

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

Executive summary:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides.

Further, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed (K-containing peptide, only) and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm (both peptides).

The following results were obtained in the DPRA:

The test substance was soluble in acetone at a concentration of 100 mM. The samples of the test substance and the peptide stock solutions were emulsions at the time of preparation and after 24 hours.

No co-elution of test substance and peptides was present. However, for the C-peptide reaction an alternative analysis method was used.

The mean C-peptide depletion, caused by the test substance was determined to be -2.27%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.40%.

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov - Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015

Deviations:

yes

Remarks:

Cell viability assay: 2 hours incubation with MTT reagent

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE; batch No.: EXP-18/11/346
- Purity test date: 18/11/2015
- Log Kow: 3.94 (calculated); 4.8 at 23°C and pH 6.0 (measured)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared within 4 hours of application as a 100x higher concentration than the highest test concentration followed by serial 1:1.2 dilution in a master plate. The test substance was further diluted 1:25 in culture medium (DMEM + 1% FBS) to obtain 4x concentrations (dilution plate).
- Visual inspection of each dilution step was performed.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in DMSO followed by dilution in culture medium (DMEM + 1% FBS)

Details on the study design:

TEST SYSTEM
- Cell line: LuSens (human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen)

CONTROLS
- Vehicle control: 1% DMSO in culture medium (DMEM + 1% FBS)
- Positive control: ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 90.8 µM (= 18 µg/ml) in 1% DMSO in culture medium (DMEM + 1% FBS)
- Negative control: DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 µM (= 450 µg/ml) in 1% DMSO in culture medium (DMEM + 1% FBS)
- Blank control: culture medium (DMEM + 1% FBS) without cells
- Basal control: culture medium (DMEM + 1% FBS) with cells

VEHICLE
- Vehicle: DMSO
- Reason for the vehicle: The test substance was soluble in DMSO.

SELECTION OF CONCENTRATIONS
- Cells were exposed to several concentrations of the test-substance preparation (0.5 µM up to 2003 µM corresponding to final test substance concentrations of 0.5 µM up to 2000 µM taking the purity/contents of 99.9% into account) and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve to be 60 µM (one pre-test) and 80 µM (further pre-test).

TEST CONCENTRATIONS
138; 115; 96; 80; 67; 55; 46; 39 µM

EXPERIMENTAL PROCEDURE
- No. of replicates: 2 independent experiments with 3 replicates in each case
- Seeding of cells: Cells were seeded in white (for luciferase assay) and clear (for cell viability assay) 96-well microtiter plates (120 µL of 0.83 x 10^5 cells/mL cell suspensions) in culture medium (DMEM + 10% FBS) and incubated for 24 hours.
- Application of test substance: After cell adaption for 24 hours, cell culture medium was exchanged by DMEM + 1% FBS. The test substance preparations (4x concentrations) were applied in a ratio of 1:4 (50 µL) to the cells (final DMSO concentration in the test medium = 1%). For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.
- Exposure duration: 48 hours under standard culture conditions (plates were sealed with semi-permeable plate sealers to prevent evaporation of the test substance)
- Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.
- Luciferase assay: After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Subsequently 200 µL of One-/Steady-Glo-preparation (= 100 µL One-/Steady-Glo- Mix and 100 µL PBS (without Ca2+/Mg2+)) per well were added and cells shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.
- Determination of cell viability: Cell culture medium was aspirated from all wells. Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and culture medium (DMEM + 1% FBS)) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

DATA EVALUATION (for details on formulas see "Any other information on materials and methods")
CV75 calculation:
- The CV75-value (relative survival rate) is calculated by linear regression. This value is the substance concentration at which relative cell viability is 75% compared to the vehicle control.
Cell viability:
- From the 3 independent replicates a mean is calculated.
Luciferase fold induction:
- From the 3 independent replicates a mean is calculated.

STATISTICAL ANALYSES
- For the statistical evaluation of luciferase fold-induction the Welch t-test (one-sided) was used.

EVALUATION CRITERIA
- A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least two consecutive concentrations of two independent experiments.
- A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 µM if molecular weight is applicable or 2000 µg/mL if molecular weight is not applicable) or maximum applicable concentration or up to the cytotoxicity limit (at least one concentration displaying viability below 70%).
- To be relevant for evaluation, the cell viability must be more than 70% in at least three tested concentrations of an experiment.

ACCEPTANCE CRITERIA
- A tested concentration is not further evaluated when relative viability is less than 70%.
- The cell viability of vehicle control cells must yield at least 85%.
- The mean of the positive control EGDMA should achieve ≥2.50 fold-induction and the mean of the LA <1.50 and the mean of the viability must be ≥70%.
- The CV [%] of the luminescence in the vehicle control wells for each plate should be below 20%.
- The mean of the basal expression of the cells must be <1.50 fold-induction as compared to the solvent control.
- Positive, negative and vehicle control data should lie within the range of the historic data.

Parameter:

other: luciferase fold induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see tab. 2

Table 1: Summary of main experiments. 2 valid and evaluable experiments (4th and 5th experiment) were performed

Concentration(testsubstance) [µM]	fold induction	4^thexperiment rel. viability [%]		t-test		Concentration (test substance) [µM]	fold induction	5^thexperiment rel. viability [%]		t-test
	mean	mean	p-value		markers		mean	mean	p-value		markers
39	1.28	103	0.004		**	39	0.99	96	0.455		n.s.
46	1.33	100	0.018		*	46	1.03	96	0.295		n.s.
55	1.38	106	0.002		**	55	1.16	99	0.128		n.s.
67	1.64	105	0.041		*	67	1.17	98	0.100		n.s.
80	1.06	44	0.367		n.s.	80	1.31	85	0.000		**
96	0.88	19	0.237		n.s.	96	1.27	36	0.083		n.s.
115	0.38	1	0.027		*	115	1.27	7	0.164		n.s.
138	0.00	-2	0.000		**	138	0.02	0	0.000		**
VC	1.00	100	-		-	VC	1.00	100	-		-
EGDMA 90.8 µM	4.42	86	0.000		**	EGDMA 90.8 µM	3.11	75	0.000		**
LA 5000 µM	1.05	104	0.225		n.s.	LA 5000 µM	1.09	95	0.180		n.s.

Table 2: Historic control data of LuSens. Data (not including present study)

Negative Control (LA 450 µg/mL)	fold induction	rel. viability [%]
Min	0.71	76
Max	1.25	140
Mean	0.95	104
SD	0.10	10
n	258

Positive Control (EGDMA 18 µg/mL)	fold induction	rel. viability [%]
Min	3.01	70
Max	10.38	135
Mean	6.01	91
SD	1.58	14
n	258

Vehicle Control (1% DMSO)	fold induction	rel. viability [%]
Min	1.00	100
Max	1.00	100
Mean	1.00	100
SD	0.00	0
n	258

Basal expression	fold induction	rel. viability [%]
Min	0.62	98
Max	1.57	186
Mean	0.99	155
SD	0.15	16
n	258

Interpretation of results:

GHS criteria not met

Conclusions:

After 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

Executive summary:

The keratinocyte activating potential of the test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer.

In order to determine the concentrations suitable for the main experiment pre-tests (non-GLP) were performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve.

In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed.

At concentrations used in the main experiment the test substance was soluble in DMSO (100 x stock preparations) and in 1% DMSO in culture medium (final concentrations). No precipitates were noticed in any concentration after 48 hours. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable due to the overall negative result of the study.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OASIS TIMES v2.27.19.13

2. MODEL (incl. version number)
Skin sensitization with autoxidation; v. 21.26

3. SMILES IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(C)Cc1ccccc1

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: In vivo: skin sensitization
- Unambiguous algorithm: refer to QMRF
- Defined domain of applicability:
1. General parametric requirements - includes ranges of variation of log KOW and MW. It specifies in the domain only those chemicals that fall in the range of variation of the MW and log Kow defined on the bases of the correctly predicted training set chemicals. This layer of the domain is applied only on parent chemicals.
2. Structural domain - it is represented by list of atom - centered fragments extracted from the chemicals in the training set. The training chemicals were split into two subsets: chemicals correctly predicted by the model and incorrectly predicted
chemicals. These two subsets of chemicals were used to extract characteristics determining the "good" and "bad" space of the domain. Extracted characteristics were split into three categories: unique characteristics of correct and incorrect chemicals (presented only in one of the subsets) and fuzzy characteristics presented in both subsets of chemicals. Structural domain is applied on parent chemicals, only.
3. Mechanistic domain - in SS model it includes: Interpolation space: this stage of the applicability domain of the model holds only for chemicals for which an additional COREPA model is required. It estimates the position of the target chemicals in the population density plot built in the parametric space defined by the explanatory variables of the model by making use the training set chemicals. Currently, the accepted threshold of population density is 10%.
The mechanistic domain is applied on the parent structures and on their metabolites.

- Appropriate measures of goodness-of-fit and robustness and predictivity:
External Validation: For substances in the applicability domain, a predictivity of 100% was found for 100 industrial chemicals for the distinction of non-sensitizers versus sensitizers of GHS Category 1. The evaluation has been published in W. Teubner, A. Mehling, P.X. Schuster, K.Guth, B. A. Worth, J. Burton, B. van Rawenzwaay, R. Landsiedel: Computer models versus reality: How well do in silico models currently predict the sensitization potential of a substance, Regulatory Toxicology and Pharmacology 67 (2013) 468-485

Statistics for goodness-of-fit: For 875 chemicals, the TIMES-SS model was able to predict correctly 90% of the strong sensitizers, 55% of the weak sensitizers and 77% of the non-sensitizers, i.e., an overall performance of 78 %. Sensitivity: 78 %, Specificity: 77 %

- Mechanistic interpretation:
The TIMES-SS (Tissue Metabolism Simulator for skin sensitization) model integrates a simulator of skin metabolism together with a number of “local” QSAR models for assessing the reactivity of specific alerts. A skin metabolism simulator was developed based on empirical and theoretical knowledge (not enough reported observed skin metabolism data). The transformation probabilities (defining the priority of their execution) were parameterized to reproduce skin sensitization data. The simulator comprises of about 420 transformations, which can be divided into four main types: abiotic transformations, covalent interaction with proteins, Phase I and Phase II reactions. Autoxidation (AU) of chemical is also accounted for. Interactions with skin proteins are grouped into three types: leading to strong or weak skin
sensitization effect and interactions requiring QSAR models to quantify the potency of sensitization of the alerting groups. The QSAR models were developed by the COmmon PAttern Recognition (COREPA) approach [3]. The skin sensitization model predicts skin sensitization effect in three classes: strong, weak and non-sensitizers.
Reliability of alerts in the TIMES-SS model has been also evaluated to provide transparent mechanistic reasoning for predicting sensitization potential. Alert performance was defined as the ratio between the number of correct (positive and negative) predictions and the total number of chemicals within the local training set that triggered the alert. The alert performance was assessed based on the predictions on parents, autoxidation products simulated by the external AU simulator and metabolites as simulated by the skin metabolism simulator embedded in TIMES-SS model. Four different categories of reliability were defined:
High reliability – alert performance higher than 60% and more than 5 chemical in local (transformation/alert) training set
Low reliability – performance less than 60% and more than 5 chemicals in training set
Undetermined reliability – less than 5 chemicals in training set
Undetermined (theoretical) – there are no chemicals supporting the alert in the local training set

5. APPLICABILITY DOMAIN
- Descriptor domain:
Log(Kow): range = [ -13.2 .. 15.4 ]
calculated: 3.94 (In domain)
MOL._WEIGHT: range = [ 30 .. 738 ]Da
calculated: 134Da (In domain)
--> Conclusion: The chemical fulfils the general properties requirements.

- Structural fragment domain: The following ACF are identified: Fragments in correctly predicted training chemicals – 100.00%, Fragments in non-correctly predicted training chemicals – 0.00%, Fragments not present in the training chemicals – 0.00%
--> Conclusion: The chemical is in the interpolation structural space

- Mechanistic domain: Interpolation space
- Similarity with analogues in the training set: not reported

6. ADEQUACY OF THE RESULT
The substance falls in the applicability domain of the model. The model was found to give reliable predictions for industrial chemicals. It is therefore considered to be acceptable for REACH.

The substance is considered to be non skin seniziting.

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs R.6, May/July 2008

Principles of method if other than guideline:

TIMES-SS v.2.27.19.13 - Skin sensitization with autoxidation v.21.26 (structure-toxicity and structure-metabolism relationships)

GLP compliance:

no

Key result

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

The registrant considers this predication as valid because TIMES-SS was validated with 100 substances from the registrant's portfolio (Teubner et al., Regulatory Toxicology and Pharmacology 67 (2013) 468–485). All predictions that fullfilled all domain requirements were correct (Specificity 100%).

The QSAR program calculated a negative sensitization potential of the test substance. The substance is in domain of the system.

Reference 4

Endpoint:

skin sensitisation: in chemico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: results derived from a (Q)SAR model using non-validated data and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
QSAR Toolbox 4.1
Database version: 4.1

2. MODEL
Skin sensitization

3. SMILES IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(C)Cc1ccccc1

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: In vivo: skin sensitization
- Unambiguous algorithm: refer to QMRF/QPRF)
- Database used: ECHA CHEM
- Appropriate measures of goodness-of-fit and robustness and predictivity: The prediction is based on 6 values, 5 of them (83,3%) equal to predicted value Prediction confidence is measured by the p-value: 0,109
- Mechanistic interpretation: not provided

5. APPLICABILITY DOMAIN
- Descriptor domain:

log Kow: from 3.09 to 4.39 --> target chemical is in domain

Protein binding by OASIS (primary grouping) --> target chemical is in domain

Organic functional groups (subcategorization) --> target chemical is in domain

Chemical elements (subcategorization) --> target chemical is in domain

Organic functional groups (nested) (subcategorization) --> target chemical is in domain

Structure similarity (subcategorization) --> target chemical is in domain

6. ADEQUACY OF THE RESULT
The substance falls in the applicability domain of the model.

The substance is considered to be non skin seniziting.

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs R.6, May/July 2008

Principles of method if other than guideline:

QSAR Toolbox 4.1 - Skin sensitization
Database version: 4.1

GLP compliance:

no

Remarks on result:

no indication of skin sensitisation

The substance is considered a non sensitizer based on read across to the following substances:

CAS Number	Substance Name
105-05-5	p-Diethylbenzene
2049-95-8	tert-Amylbenzene
98-82-8	Cumeme
95-47-6	2-Xylene
108-38-3	m-Xylene

Interpretation of results:

GHS criteria not met

Conclusions:

The QSAR program calculated a negative sensitization potential of the test substance based on read across analysis using the nearest 5 neighbours. The substance is in domain of the system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

To assess the skin sensitizing potential of the test substance an in vitro skin sensitization turnkey testing strategy was conducted. The following tests were used:

DPRA:

In order to assess the reactivity towards peptides, synthetic cysteine (C)- or lysine (K)-containing peptides were incubated with the testsubstance for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UVdetection at 220 nm according to the OECD guideline 442C.

Therefore, the test substance was dissolved at a 100 mM concentration in acetonitrile and three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide).

The test substance caused a mean C-peptide depletion of -2.27% and a mean K-peptide depletion of -0.40%.

The mean peptide depletion was calculated to be 0.00% as negative values were considered to be "zero".

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

LuSens:

In order to assess the keratinocyte activating potential, a luciferase reporter cell line (LuSens cells) was incubated with the test substance for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer according to the OECD guidline 442D.

Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable due to the overall negative result of the study.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

Examination of the test substance in two different non-animal methods addressing different key events of the skin sensitization Adverse Outcome Pathway resulted in one negative and one inconclusive result in the LuSens and DPRA, respectively. A further test (h-CLAT) determining the potential of the test substance to activate dendritic cells was not conducted, because according to the OECD guideline the substance´s log KOW which is > 3.5 would not allow a valid evaluation of negative test results.

Applying the Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification the skin sensitization potential of the test substance cannot be conclusively evaluated based on the results of this in vitro skin sensitization turnkey testing strategy. However, the results indicate, that the test substance is predicted to be not sensitizing, because the molecular initiating event of protein binding also occurs in the cell-based LuSens, which revealed a valid negative result.

To confirm this conclusion a quantitative structure-activity relationship (QSAR) system for the estimation of the skin sensitization potency that incorporates skin metabolism and considers the potential of parent chemicals and/or their activated metabolites to react with skin proteins was used. Two QSAR programs (TIMES-SS and QSAR Toolbox) calculated a negative sensitization potential of the test substance. The test substance was in domain of the systems. The registrant considers this prediction as valid, because TIMES-SS was validated from the registrant´s portfolio (Teubner et al., Regulatory Toxicology and Pharmaclogy 67 (2013) 468-485).

Based on this weight of evidence approach consisting of in vitro tests and QSAR programs, that uniformly predict the test substance as a non-skin sensitizer, the test substance is judged not to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.