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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2019-2-11 to 2019-2-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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:
2015
Deviations:
no
GLP compliance:
yes
Type of study:
other: Direct Peptide Reactivity Assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-dihydro-1,4-benzodioxine-2-carboxylic acid
EC Number:
609-276-2
Cas Number:
3663-80-7
Molecular formula:
C9H8O4
IUPAC Name:
2,3-dihydro-1,4-benzodioxine-2-carboxylic acid
Test material form:
solid
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a

In chemico test system

Details on the study design:
TEST SYSTEM
- Test system : Synthetic peptides containing cysteine (SPCC) or synthetic peptides containing lysine (SPCL)
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH

EXPERIMENTAL DESIGN
- Test Item Preparation: 28.50 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1582 µL ACN after vortex mixing to obtain a 100 mM solution.
- 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 10.5 mg of SPCC in 20.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
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.
- 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 10.2 mg of SPCL in 19.69 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
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.
- 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 23.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
- HPLC Analysis: SPCC and SPCL peak areas in the samples were measured by HPLC.
- Other measurement: Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

Results and discussion

Positive control results:
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 91.6% ± 0.1%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 47.9% ± 4.9%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Cysteine Reactivity Assay
Parameter:
other: SPCC depletion%
Value:
0
Positive controls validity:
valid
Remarks on result:
other: the mean Percent SPCC Depletion for the test item
Key result
Run / experiment:
other: Lysine Reactivity Assay
Parameter:
other: SPCL depletion%
Positive controls validity:
valid
Remarks on result:
other: the test item co-eluted with SPCL and therefore the Percent SPCL Depletion could not be calculated.
Other effects / acceptance of results:
- Precipitation: No precipitate or phase separation was observed in any of the samples for both Cysteine Reactivity Assay and Lysine Reactivity Assay.
- Test Acceptability: all acceptability criteria were met this DPRA is considered to be valid.

Any other information on results incl. tables

In the cysteine reactivity assay the test item showed 0.0% SPCC depletion while in the lysine reactivity assay the test item showed interference with SPCL. As a result, the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 prediction model.

Applicant's summary and conclusion

Interpretation of results:
other: negative
Conclusions:
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 prediction model.
Executive summary:

The objective of this study was to determine the reactivity of the test substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OCED Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)).

After incubation of the test item 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 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.

 

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 0.0% SPCC depletion while in the lysine reactivity assay the test item showed interference with SPCL. 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 prediction model.

 

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test substance was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model.