1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May 2020 - 16 Jun 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol

EC Number:

255-256-8

EC Name:

1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol

Cas Number:

41199-19-3

Molecular formula:

C13H22O

IUPAC Name:

2,5,5-trimethyl-1,2,3,4,4a,5,6,7-octahydronaphthalen-2-ol

Test material form:

liquid

In chemico test system

Details on the study design:

TEST ITEM PREPARATION
- 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), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol (MeOH) and ethanol (EtOH).
- Test item stock solutions were prepared freshly for each reactivity assay.
- The dissolution of the test item in the SPCC and SPCL assay buffers was also evaluated by diluting the test item stock solution in the buffer based incubation mixtures. For the SPCC assay, a 20-fold dilution was prepared by mixing one volume of the test item stock solution with fifteen volumes of phosphate buffer pH 7.5 and four volumes of ACN. For the SPCL assay, a 4-fold dilution was prepared by mixing one volume of the test item stock solution with three volumes of ammonium acetate buffer pH 10.2. The presence of cloudiness, precipitate and/or phase separation was evaluated by visual inspection to aid solvent selection for the main study.
- For the cysteine and lysine reactivity assay respectively 36.27 mg and 31.83 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1867 μL and 1638 μL ACN, respectively, to obtain 100 mM solutions. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

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. Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Storage: The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months.
- Incubation: The samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark andn 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 were 23.9 hours and 23.7 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
- Analysis: All samples were analyzed according to the HPLC method presented in Table 1. (See 'other information on materials and methods').
- The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2. (See 'other information on materials and methods').

POSITIVE CONTROL
Cinnamic aldehyde
- Purity: 99.1%
- Expiry of batch: 30 November 2021

DATA EVALUATION
- The concentration of SPCC or SPCL was spectrophotometrically 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: Percent Peptide Depletion= [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]x100
- 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 (see Table 3), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have a r2>0.99.
b) 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.
c) 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.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide peak 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:
a) 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.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

run/experiment 1

Parameter:

mean cystein depletion

Value:

74.1 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 1.3%

Positive controls validity:

valid

Remarks:

Mean percentage SPCC: 73.9 ±1.3%

Results 2

Key result

Run / experiment:

run/experiment 1

Parameter:

mean lysine depletion

Value:

0 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 0.9%

Positive controls validity:

valid

Remarks:

Mean percentage SPCL: 63.8 ± 0.5%

Other effects / acceptance of results:

Cysteine Reactivity Assay for the Test Item
- Preparation of a 100 mM test substance stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the test item samples. 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 211255/A-cys samples, the mean SPCC A220/A258 area ratio was 37.86. Since this was within the 33.69-41.17 range, this again indicated that there was no co-elution of the test item with SPCC.

Lysine Reactivity Assay for the Test Item
- Preparation of a 100 mM test substance stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a precipitate was observed in the CC and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide. Upon preparation as well as after incubation of the SPCC no precipitate or phase separation was observed in any of the samples. Upon preparation as well as after incubation of the SPCL test item samples, a precipitate was observed.

See Table 4 & 5 "any other information on results incl. tables" for acceptability criteria.

Any other information on results incl. tables

Table 4: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay	Cysteine reactivity assay	Lysine reactivity assay	Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.9992	>0.99	1.000
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.507 ± 0.001	0.50 ± 0.05	0.495 ± 0.001
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.501 ± 0.004	0.50 ± 0.05	0.499 ± 0.004
CV (%) for RC samples B and C	<15.0	1.3	<15.0	0.9
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	73.9	40.2-69.0	63.8
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.3	<11.6	0.5
SD of peptide depletion for the test item (%)	<14.9	1.3	<11.6	0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Table 5: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

	SPCC depletion Mean	SPCC depletion ± SD	SPCL depletion Mean		SPCL depletion ± SD	Mean of SPCC and SPCL depletion	Cysteine 1:10 / Lysine 1:50 prediction model
Test item	74.1%	±1.3%		0.0%	±0.0%	37.1%	Positive: Moderate reactivity

SD = Standard Deviation.

Applicant's summary and conclusion

Conclusions:

The substance was positive in the DPRA and was classified in the “moderate reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In an in chemico (DPRA) study, performed according to OECD guideline 442C and in accordance with GLP principles, the reactivity of the test substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined. Following incubation of the test substance with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in the prediction model. ACN was found to be an appropriate solvent to dissolve the test substance and was therefore used in this DPRA study. Cinnamic aldehyde was used as a positive control. All acceptability criteria were met and therefore, the study was considered to be valid. No precipitate or phase separation was observed in the SPCC samples before and after the incubation. Upon preparation as well as after incubation of the SPCL test item samples, a precipitate was observed. In the cysteine reactivity assay the test item showed 74.1% SPCC depletion and in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 37.1% and as a result the substance was considered to be positive in the DPRA and classified in the “moderate reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.