3β-hydroxyandrost-5-en-17-one acetate

Endpoint summary

Description of key information

Skin sensitisation:

In silico, in chemico and in vitro data are used in a weight-of-evidence approach. In addition, the Local Lymph Node Assay (LLNA) is added as key study.

- In silico: A Derek Nexus assessment did not yield an alert for skin sensitisation (“no alerts matched”) (De Vlieger, 2017).

- In chemico (OECD 442C): The test item was considered to be positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model (Rijk, 2018). However, since precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the % peptide depletion may be underestimated.

- In vitro (OECD 442D): The KeratinoSens^TM assay resulted in an inconclusive outcome, as negative results (<1.5-fold induction) were observed at test concentrations < 1000 µM (Westerink, 2018).

Taking into account the negative in silico, the positive in chemico and inconclusive in vitro results and as it is not possible to determine the potency (Cat. 1A or 1B) based on non-animal testing approaches, as required in Annex VII, section 8.3, additional in vivo testing (Local Lymph Node Assay in mice according to OECD 429) was performed to assess the potency of the test item.

- In vivo (OECD 429): In a Local Lymph Node Assay in female CBI mice the test item was not regarded as a skin sensitizer according to the recommendations made in the test guidelines - no indication that the test item elicits a SI ≥ 3 when tested up to 25% (van Sas, 2018).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-17 to 2018-02-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Paris Cedex, July 2010.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Official Journal of the European Union No. L142, May 2008, including most recent amendments.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16GB2547
- Expiration date of the lot/batch: 01 July 2018 (retest date)
- Purity/composition correction factor: 1
- Purity: 99.6% w/w (calculated as is) - HPLC

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: There was no information available regarding the solubility or stability in vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item was not applicable, since the test method required a logical concentration range rather than specific dose levels to be dosed.

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/J, inbred, SPF-Quality; Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Aproximately 10 weeks old
- Weight at study initiation: 18.7 - 23.9 grams
- Housing: Group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. On Day 6 of the main study, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 18 to 24°C: the actual daily mean temperature during the study period was 22 to 23°C
- Humidity (%): 40 to 70%: the actual daily mean relative humidity during the study period was 41 to 44%
- Air changes (per hr): At least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

PreScreen Test: 25% and 50% w/w
Main Study: 0, 2%, 10%, 25% w/w

No. of animals per dose:

Five females per group; 4 groups (including control group)

Details on study design:

Rationale vehicle selection:
The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at the testing facility. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v) (turbid solution), N,N-dimethylformamide (turbid solution), methylethylketone (turbid solution), propylene glycol (turbid solution) and dimethylsulfoxide (formulation too dry). As no difference was seen in solubility for several vehicles the vehicle used in the reliability check was selected.

PRE-SCREEN TESTS:
- Compound solubility: no data
- Irritation: No to very slight irritation was observed in any of the animals examined. White test remnants were present on the dorsal surface of the ears of the animals between Days 1 and 3, which did not hamper scoring of the ears.
- Systemic toxicity: No signs of systemic toxicity were observed in any of the animals examined
- Ear thickness measurements: Variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values for the animals treated at a concentration of 50%. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for the animals treated at a concentration of 25%, except for one ear of one animal on Day 6 only, for which the increase was 27%. Based on these results, the highest test concentration selected for the main study was a 25% concentration and ear thickness measurements were added to the main study to clarify the findings in one single pre-screen animal on Day 6.
- Erythema scores: 25% w/w group: erythema score of 0 in all animals (2 in total) during the observation period; 50 % w/w group: erythema score of 1 on day of the observation period in all animals (2 in total) and 0 in all animals from day 2 until day 6 of the observation period


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, including all amendments. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3).

Classification of results:
SI value UN-GHS 2015; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer -
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skinreaction
EC3 value ≤ 2%: sub-category 1A
EC3 value > 2%: sub-category 1B

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

No data

Positive control results:

At concentrations 5%, 10% and 25% SI values of the positive control item were 1.4, 2.2, and 3.5 respectively. An EC3 value of 19.2% was calculated using linear interpolation.
The calculated EC3 value was in the accepable range of 4.8 and 19.5%. The results of the 6 monthly reliability checks of the recent years were 13.2, 14.1, 17.3, 9.8, 17.8, 18.0, 14.7 and 13.2%
Based on the results, it was concluded that the Local Lymph Node Assay as performed in the laboratory is an appropriate model for testing contact hypersensitivity.

Parameter:

SI

Value:

1.2

Test group / Remarks:

Based on 5 animals in 2% w/w in acetone/olive oil 4:1 group

Parameter:

SI

Value:

1

Test group / Remarks:

Based on 5 animals in 10% w/w in acetone/olive oil 4:1 group

Parameter:

SI

Value:

0.8

Test group / Remarks:

Based on 5 animals in 25% w/w in acetone/olive oil 4:1 group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
0% w/w group: mean DPM ± SEM: 818 ± 109
2% w/w group: mean DPM ± SEM: 1011 ± 92
10% w/w group: mean DPM ± SEM: 843 ± 126
25 %w/w group: mean DPM ± SEM: 670 ± 85
SEM = Standard Error of the Mean

EC3 CALCULATION
not applicable for this test (SI values < 3)

CLINICAL OBSERVATIONS:
- Skin reactions/irritation:
No irritation of the ears was observed in any of the animals examined. White test item remnants were present on the dorsal surface of the ears of animals treated at a concentration of 10 and 25% on Days 2 and 3, which did not hamper scoring of the skin reactions.
- Systemic toxicity:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.
-Ear thickness:
Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for all animals. This confirms that the increase noted in the pre-screen test was an incidental finding.
- Macroscopy of the auricular lymph nodes and surrounding area:
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 25%, it was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 25%.

Based on these results, T008506 was not regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-09-13 to 2017-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: M16GB2547
- Expiration date of the lot/batch: 2018-07-01 (retest date)
- Purity (HPLC): 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), MQ/ACN (1:1, v/v), isopropanol, acetone, acetone/ACN (1:1, v/v) and dimethylsulfoxide (DMSO)/ACN (1:9, v/v).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: For the cysteine and lysine reactivity assay 51.10 mg of the test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1546 µL ACN to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved.

OTHER SPECIFICS: no correction (correction factor =1.00) was made for the purity/composition.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

TEST SYSTEM
- Test system: Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC, and 775.9 g/mol for SPCL.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Batch SPCC: 111016HS_MHe_W0417
- Batch SPCL: 220114HSDW_W0417
- Storage: The peptides were stored in the freezer (≤ 15°C) for a maximum of 6 months.

EXPERIMENTAL DESIGN
TEST ITEM PREPARATION
see details under "Specific details on test material used for the study"

PREPARATION OF SOLUTIONS FOR CYSTEINE REACTIVITY ASSAY
- SPCC stock solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC reference control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.
- SPCC calibration curve: A SPCC calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

PREPARATION OF SOLUTIONS FOR LYSINE REACTIVITY ASSAY
- SPCL stock solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL reference control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.
- SPCL calibration curve: A SPCL calibration curve was prepared as described under "Any other information on materials and methods incl. tables".
- Co-elution control, Test item and Positive control samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described under "Any other information on materials and methods incl. tables".

SAMPLE INCUBATIONS
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 22.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 11 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature.

HPLC-PDA Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following system:
System 1 (used for Cysteine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
- LC Column oven 300 (Thermo Scientific)
- Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
- Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
- HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
- Column Oven #151006 (Grace, Worms, Germany)
- Surveyor PDA detector (Thermo Scientific)

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
- The standard calibration curve had to have an r²>0.99.
- The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
- The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
- The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
- The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
- The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
- The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.
All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not have been exactly reproduced from the individual data presented.

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
% Peptide Depletion = [ 1 - (Peptide Peak Area in Replication injection at 220 nm / Mean Peptide Peak Area in Reference Controls at 220 nm) ] x 100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Positive control results:

Cysteine reactivity assay:
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde, calculated versus the mean SPCC peak area of Reference Controls C, was 71.3% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%). This was just below the lower limit of the historical positive control data range.

Lysine reactivity assay:
The Percent SPCL Depletion for the positive control cinnamic aldehyde, calculated versus the mean SPCL peak area of Reference Controls C, was 41.1% ±1.9%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%). This was just below the lower limit of the historical positive control data range.

Run / experiment:

other: Run 1 / mean of SPCC and SPCL depletion

Parameter:

other: Mean of SPCC and SPCL depletion (%)

Value:

8.5

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: Since precipitation of the test item was observed in the cysteine as well as in the lysine reactivity assay, the % peptide depletion for both SPCC and SPCL might be underestimated

Run / experiment:

other: Run 1 / mean of 3 replicates

Parameter:

other: % SPCC depletion

Value:

16.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Since precipitation of the test item was observed in the cysteine as well as in the lysine reactivity assay, the % peptide depletion for both SPCC and SPCL might be underestimated

Run / experiment:

other: Run 1 / mean of 3 replicates

Parameter:

other: % SPCL depletion

Value:

0.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Since precipitation of the test item was observed in the cysteine as well as in the lysine reactivity assay, the % peptide depletion for both SPCC and SPCL might be underestimated

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The DPRA assay was successfully validated at the laboratory and can be used to support the discrimination between sensitisers and non-sensitisers.

ACCEPTANCE OF RESULTS - Cysteine reactivity assay
- Correlation coefficient (r²) standard calibration curve: 0.993 was within the acceptance criteria (r²>0.99) (SPCC standard calibration curve accepted)
- Mean peptide concentration Reference Control A samples: 0.512 ± 0.006 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.525 mM ± 0.012 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A and C were both within the acceptance criteria. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 2.7% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 71.3% was within the acceptance range of 60.8% to100%
- SD of peptide depletion cinnamic aldehyde: 0.9% was below the maximum (SD <14.9%)

ACCEPTANCE OF RESULTS - Lysine reactivity assay
- Correlation coefficient (r²) standard calibration curve: 0.997 was within the acceptance criteria (r²>0.99) (SPCL standard calibration curve accepted)
- Mean peptide concentration Reference Control A samples: 0.503 ± 0.007 mM was within the acceptance criteria of 0.50 ± 0.05 mM
- Mean peptide concentration Reference Control C samples: 0.506 ± 0.008 mM was within the acceptance criteria of 0.50 ± 0.05 mM
The mean of Reference Control Samples A and C were both within the acceptance criteria. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
- Coefficient of Variation (CV) for Reference Control samples B and C: 1.0% was within the acceptance criteria (CV<15.0%) (confirms stability of the HPLC run over time)
- Mean peptide depletion cinnamic aldehyde: 41.1% was within the acceptance range of 40.2% to 69.0%
- SD of peptide depletion cinnamic aldehyde: 1.9% was below the maximum (SD <11.6%)

Solubility assessment:

At a concentration of 100 mM, the test item was not soluble (precipitation) in MQ, MQ/ACN (1:1, v/v), but was soluble in ACN, isopropanol, acetone, acetone/ACN (1:1, v/v) and DMSO/ACN (1:9, v/v). Since ACN is the preferred solvent to be used in the DRPA, this solvent was used to dissolve the test item in this DPRA study.

Results cysteine reactivity assay for the test item:

Preparation of a 100 mM test item stock solution in ACN showed that the test item was dissolved completely. Upon preparation as well as after incubation a precipitate was observed in the co-elution control (CC) and test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the test item samples, the mean SPCC A220/A258 area ratio was 18.71. Since this was within the 16.11 -19.69 range (mean A220/A258 ratio ± 10% range of Reference controls A, B and C). This again indicated that there was no co-elution of the test item with SPCC.

Results lysine reactivity assay for the test item:

Preparation of a 100 mM test item stock solution in ACN showed that the test item was dissolved completely. Upon preparation as well as after incubation a precipitate was observed in the co-elution control (CC) and test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the test item samples, the mean SPCL A220/A258 area ratio was 13.91. Since this was within the 12.50 -15.27 range (mean A220/A258 ratio ± 10% range of Reference controls A, B and C). This again indicated that there was no co-elution of the test item with SPCL.

SPCC and SPCL depletion, DPRA prediction and reactivity classification:

SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
16.1%	± 5.1%	0.9%	± 0.8%	8.5%	Positive: Low reactivity

SD = Standard Deviation                                 

Historical control data for DPRA studies

	Positive control - Cinnamic aldehyde
	SPCC depletion	SPCL depletion
Range	71.8 - 78.1%	43.5 - 65.2%
Mean	74.8%	59.1%
SD	1.7%	5.2%
n	31	31

SD = Standard Deviation, n = Number of observations

The above mentioned historical control data were collected over the period of January 2017 to August 2017.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.
The test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, the % peptide depletion may be underestimated.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-10-16 to 2017-11-03

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: M16GB2547
- Expiration date of the lot/batch: 01 July 2018 (retest date)
- Purity (HPLC): 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test with dimethyl sulfoxide (DMSO) was performed. The test item was suspended in DMSO to a final concentration of 200 mM (white suspension). The suspension was vortexed and sonicated (20 minutes with a maximum temperature of 30.0 °C) to get a homogeneous suspension. The 100-fold dilution in DMEM of the 200 mM stock showed moderate precipitation and was therefore not suitable to test. Next a 100 mM stock in DMSO was prepared (white suspension). The 100-fold dilution in DMEM of 100 mM showed moderate precipitation and was therefore not suitable to test.
At 50, 25 and 12.5 mM the test item dissolved in DMSO. The 100-fold dilution of the 50 and 25 mM DMSO stocks showed slight precipitation (final concentration of 500 and 250 μM) and the 100-fold dilution of the 12.5 mM DMSO stock showed no precipitation (final concentration 125 μM). A concentration of 500 μM was selected as highest concentration for the main assay (highest feasible concentration based on solubility).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 50 mM. The stock was vortexed and sonicated (11 minutes with a maximum temperature of 35°C in experiment 1 and for 24 minutes with a maximum of 28.0°C in experiment 2) to dissolve the test item. From the 50 mM stock 11 spike solutions in DMSO were prepared (2-fold dilution series).

OTHER SPECIFICS: No correction (correction factor = 1.00) was necessary for the purity/composition of the test item

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

CONTROLS
- solvent control: 1% DMSO in exposure medium
- positive control: Ethylene dimethacrylate glycol

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- A solubility test with dimethyl sulfoxide (DMSO) was performed as described in Specific details on test material used for this study
- In the main experiments a 50 mM stock solution in DMSO was prepared. The stock was vortexed and sonicated (11 minutes with a maximum temperature of 35°C in experiment 1 and for 24 minutes with a maximum of 28.0°C in experiment 2) to dissolve the test item. From the 50 mM stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. Precipitation was observed at the start and end of the incubation period in the 96-well plates at the final concentration of 500 μM.
- Test item concentrations were used within 2 hours after preparation. Any residual volumes were discarded.

PREPARATION OF THE POSITIVE CONTROL
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol, for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

SOLVENT CONTROL
The solvent control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

BLANK
On each plate three blank wells were tested (no cells and no treatment).

TEST SYSTEM
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSensTM cell line). The KeratinoSensTM cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells were propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum of 25 passages from the stock (p+25) and are employed for routine testing using the appropriate maintenance medium. Once a year the cell line is checked for infection with a mycoplasma detection test.

CELL CULTURE
- Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
- Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).
- Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

ENVIRONMENTAL CONDITIONS
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 80 - 99 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. The temporary deviations, from the temperature (0.4°C) occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

EXPERIMENTAL DESIGN
- Two experiments were conducted
- Subculturing: Cells were subcultured upon reaching 80-90% confluency in maintenance medium. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (p+25).
- Plating of cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, one white 96-well plate (3 replicates per concentration) was used for the luciferase activity measurements, and one parallel 96-well transparant plate (3 replicates per concentration) was used for the MTT cell viability assay. The cells were incubated for 24 ± 1 hours in the incubator. The passage number used was p+15 in experiment 1 and p+3 in experiment 2.
- Treatment of cells: The medium was removed and replaced with fresh culture medium (150 μL exposure medium) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. Initially, experiment 2 did not pass all the acceptability criteria of the positive control (not reported) and therefore this part of the study (including the test item, positive and negative control) was repeated. In total 2 valid experiments were conducted.
- Luciferase activity measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together at room temperature. Prior to addition to the cells the Steady-Glo Luciferase substrate was mixed 1:1 with exposure medium. The assay plates were removed from the incubator and the medium was removed. Then 100 μL of PBS was added to rinse the cells. After removing the PBS, 200 µL of the Steady-Glo Luciferase substrate solution was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
- Cytotoxicity assessment: For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh DMEM-glutamax containing 5 mg/mL MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the average induction in the three replicates for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
The coefficient of variation is calculated with the following formula:
Coefficient of Variation = [(Standard deviation / Mean luminescence reading) x 100%]

DATA EVALUATION AND STATISTICAL PROCEDURES
The following parameters were calculated in the KeratinoSensTM test method:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained.
- The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
Imax, EC1.5, IC50 and IC30 were calculated based on the equations stated in the OECD Guideline 442D.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration. ToxRat Professional will be used for statistical analysis of the data.
Additional points for data analysis:
- A graph was produced to help visually check the data. If no clear dose response curve was observed, or if the dose-response curve obtained was biphasic (i.e. crossing the threshold of 1.5 twice), the experiment was repeated to verify whether this was specific to the test chemical or due to an experimental artefact. In case the biphasic response was reproducible in an independent experiment, the lower EC1.5 value (the concentration when the threshold of 1.5 is crossed the first time) should be reported.
- In the rare cases where a statistically non-significant induction above 1.5 fold was observed followed by a higher concentration with a statistically significant induction, results from this repetition were only considered as valid and positive if the statistically significant induction above the threshold of 1.5 was obtained for a non-cytotoxic concentration.
- Finally, for test chemicals generating a 1.5 fold or higher induction already at the lowest test concentration of for example 0.977 μM, the EC1.5 value of <0.977 was set based on visual inspection of the dose-response curve.

DATA INTERPRETATION
A minimum of two experiments was conducted, in case of two not concordant results, a third experiment was performed. A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM.
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations < 1000 µM should be considered as inconclusive

Positive control results:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (120 μM and 57 μM in experiment 1 and 2, respectively). A clear dose response was observed and the induction at 250 μM was 1.88-fold and 2.52-fold in experiment 1 and 2,respectively. In the first experiment the EC1.5 fell outside the historical control data (> 119.2 μM, >Mean+2xSD), however it did meet the acceptation criteria as it was situated between 5 and 125 μM. Although in the first experiment the Imax did not meet the 2-fold increase acceptability criterium (1.88-fold), a clear doseresponse was observed. Therefore the study outcome was not affected and the first experiment was as well considered valid.

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.04

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No EC1.5 could be calculated

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No EC1.5 could be calculated

Run / experiment:

other: Experiment 1

Parameter:

other: IC30 (in µM)

Value:

249

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 2

Parameter:

other: IC30 (in µM)

Value:

176

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 1

Parameter:

other: IC50 (in µM)

Value:

321

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 2

Parameter:

other: IC50 (in µM)

Value:

219

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The KeratinoSensTM assay was successfully implemented and validated, and lab proficiency has been shown by obtaining the expected KeratinoSensTM prediction for the 10 proficiency chemicals that are described in the OECD 442D guideline.

ACCEPTANCE OF RESULTS:
Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (120 μM and 57 μM in experiment 1 and 2, respectively). A clear dose response was observed and
the induction at 250 μM was 1.88-fold and 2.52-fold in experiment 1 and 2, respectively). In the first experiment the EC1.5 fell outside the historical control data (> 119.2 μM, >Mean+2xSD), however it did meet the acceptation criteria as it was situated between 5 and 125 μM. Although in the first experiment the Imax did not meet the 2-fold increase acceptability criterium (1.88-fold), a clear doseresponse was observed. Therefore the study outcome was not affected and the first experiment was as well considered valid.
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (3.1% and 4.6% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Test item results

Experiment 1:

- Precipitation was observed at the start and end of the incubation period in the 96-well plates at the top dose level of 500 μM.

- The test item showed toxicity. The calculated IC30 was 249 μM and the calculated IC50 was 321 µM.

- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.04 and therefore no EC1.5 could be calculated.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 1.88 and the EC1.5 120 μM.

Experiment 2:

- Precipitation was observed at the start of the incubation period in the 96-well plates at at the top dose level of 500 µM.

-The test item showed toxicity. The calculated IC30 was 176 μM and the calculated IC50 was 219 µM.

- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.11 and therefore no EC1.5 could be calculated.

- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.52 and the EC1.5 57 μM.

Historical Control Data for the KeratinoSensTM Studies:

	Positive control
	EC1.5 (µM)	Imax (µM)
Range	6.3 - 119.2	1.8 - 52.7
Mean	42.9	4.8
SD	28.3	6.8
n	81	77

SD = Standard Deviation; n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2016 to July 2017.

Interpretation of results:

other: inconclusive

Remarks:

negative results (<1.5- fold induction) were observed at test concentrations < 1000 μM

Conclusions:

In conclusion, the test item is classified as inconclusive (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described.

Reference 4

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

08 September 2017

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
Derek Nexus

2. MODEL (incl. version number)
Derek Nexus: 5.0.2, Nexus: 2.1.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
O(C(C)=O)[C@@H]4CC=3[C@]([C@@]1([C@]([C@]2([C@](CC1)(C(CC2)=O)C)[H])(CC=3)[H])[H])(CC4)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
see attached QMRF
Q13-34-36-315
The corresponding QMRF named “Derek for Windows - Skin sensitization” has been downloaded from the JRC QSAR model Database.

5. APPLICABILITY DOMAIN (see Derek report in field 'Attached background material')
No alerts matched

6. ADEQUACY OF THE RESULT (see Derek report in field 'Attached background material')
not applicable

Principles of method if other than guideline:

- Software tool(s) used including version: Derek Nexus: 5.0.2; Nexus: 2.1.1
- Model(s) used: Knowledge Base: Derek KB 2015 2.0
- Model description: see QMRF in field 'Attached justification'
- Justification of QSAR prediction: the QSAR prediction for skin sensitisation is used as part of the weight-of-evidence approach to cover the information requirements for this endpoint. The justification is further elaborated in the weight-of-evidence justification attached to the skin sensitisation endpoint summary in this dossier.

Specific details on test material used for the study:

SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: O(C(C)=O)[C@@H]4CC=3[C@]([C@@]1([C@]([C@]2([C@](CC1)(C(CC2)=O)C)[H])(CC=3)[H])[H])(CC4)C
- Average Mol Mass: 330.46
- Exact Mol Mass: 330.2195
- Log Kp: -1.89
- Log P: 4.01
- Composition / purity: other information: not applicable for in silico study

Parameter:

other: no prediction on EC3

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Reasoning Summary

No alerts matched

Interpretation of results:

GHS criteria not met

Conclusions:

No alerts were matched. Therefore, there is no indication of skin sensitisation using Derek Nexus v5.0.2.

Reference 5

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In silico, in chemico and in vitro data are used in a weight-of-evidence approach. The studies are discussed in detail in the Weight-of-Evidence justification attached to this endpoint summary.

In addition, the Local Lymph Node Assay (LLNA) is added as key study.

Test item concentrations selected for the main study were based on the results of a pre-screen test. 

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 2, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

Three days after the last exposure, all animals were injected with³H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation of the ears was observed in any of the animals examined. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 10 and 25% were 1011, 843 and 670 DPM, respectively. The mean DPM/animal value for the vehicle control group was 818 DPM. The SI values calculated for the item concentrations 2, 10 and 25% were 1.2, 1.0 and 0.8, respectively.

Since there was no indication that the test item elicits a SI≥3 when tested up to 25%, the test item was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 25%.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation:

Taking into account the negative in silico, the positive in chemico and inconclusive in vitro results and as it is not possible to determine the potency (Cat. 1A or 1B) based on non-animal testing approaches, as required in Annex VII, section 8.3, additional in vivo testing (Local Lymph Node Assay in mice according to OECD 429) was performed to assess the potency of the test item.

Based on the results of the Local Lymph Node Assay in female CBI mice the test item is not classified a skin sensitizer no indication that the test item elicits a SI ≥ 3 when tested up to 25% (van Sas, 2018).