3-octoxypropane-1,2-diol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 Sep 2019 to 06 sep 2019

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:

2019

Report date:

2019

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)

Justification for non-LLNA method:

A 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 one of the non-animal tests to be performed as part of weight of evidence.

Test material

Test material information

Constituent 1

Reference substance name:

3-(octyloxy)propane-1,2-diol

Cas Number:

10438-94-5

Molecular formula:

C11H24O3

IUPAC Name:

3-(octyloxy)propane-1,2-diol

Test material form:

liquid

Details on test material:

Storage Conditions: At room temperature protected from light container flushed with nitrogen.

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 solvent was evaluated: acetonitrile (ACN).
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 34.78 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1702 µL ACN after vortex mixing 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. 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
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.
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 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 24.2 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence 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%
- Batch: MKCB9907
- 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))]×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 (see Table 3 `other information on materials and method')
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 below), 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 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:

other: Cysteine Reactivity Assay

Parameter:

other: Mean Percentage SPCC depletion

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV Between reference controls: 0.6%

Positive controls validity:

valid

Remarks:

mean percentage SPCC: 71.1% ± 0.2%.

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: Mean Percentage SPCL depletion

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 0.4%

Positive controls validity:

valid

Remarks:

Mean percentage SPCL: 62.3% ± 3.4%

Other effects / acceptance of results:

- Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.
- In the cysteine reactivity assay the test item showed 0.8% SPCC depletion while in the lysine reactivity assay the test item showed 0.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.6% and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Any other information on results incl. tables

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	1.000	>0.99	1.000
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.506 ± 0.002	0.50 ± 0.05	0.502 ± 0.001
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.507 ± 0.003	0.50 ± 0.05	0.501 ± 0.002
CV (%) for RC samples B and C	<15.0	0.6	<15.0	0.4
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	71.1	40.2-69.0	62.3
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.2	<11.6	3.4
SD of peptide depletion for the test item (%)	<14.9	0.2	<11.6	0.2

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

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forthe Test Item

Test item	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
Saskine ™ 80	0.8%	±0.2%	0.4%	±0.2%	0.6%	Negative: No or minimal reactivity

SD = Standard Deviation.

Applicant's summary and conclusion

Interpretation of results:

other: study is part of a weight of evidence approach and is not used for classification on its own

Conclusions:

In a DPRA performed according to OECD TG 442C the mean of the SPCC and SPCL depletion was 0.6% and the substance is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

A DPRA study was performed according to the OECD TG 442C and in accordance with GLP principles. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. The relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples, the CV for RC samples, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA. Since all acceptability criteria were met this DPRA is considered to be valid. No coelution of the test item with SPCC or SPCL was observed.

In the cysteine reactivity assay the test item showed 0.8% SPCC depletion while in the lysine reactivity assay the test item showed 0.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.6% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.