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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:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethyl phosphate
EC Number:
208-144-8
EC Name:
Trimethyl phosphate
Cas Number:
512-56-1
Molecular formula:
C3H9O4P
IUPAC Name:
trimethyl phosphate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No. of test material: Sigma Aldrich, Saint Louis, USA; MKBX2207V
- Expiration date of the batch: 26/06/2022
- Purity test date: 97 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: yes
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was prepared at 100 mM in acetonitrile and the positive control was prepared at 100 mM in acetonitrile.

In chemico test system

Details on the study design:
Skin sensitisation (In chemico test system) - Details on study design:
The test item was prepared at 100 mM in acetonitrile and the positive control (100 mM cinnamaldehyde (i. e. 5 or 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine)) was prepared at 100 mM in acetonitrile.
They were incubated in excess with the peptides at 1:10 and 1:50 ratio for cysteine and lysine peptides respectively.
Each sample was tested 3 times from 3 independent solutions.
3 references made with 0.500 mM* peptides solutions in acetonitrile or in the test item solvent were included in the analysis:
- Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy,
- Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time,
- Reference control C: prepared with acetonitrile, the test item and the positive control solvent in order to check its influence on the peptide stability.

Preparation of the test item and positive control:
- 36 µI was distributed in a glass vial in order to prepare, right for use, 3 ml of a limpid 100 mM solution.
- 38.2 µI of the positive control were distributed in a 5 ml glass vial in order to prepare, right before use, 3 ml of a limpid 100 mM solution.

Preparation of the sample for the test:
Samples were dissolved immediately before use (100 mM positive control solution was kept for the 2 runs). Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively. All the replicates were prepared with the same peptide stock solutions.
The vials were capped and mixed carefully avoiding bubbling, then placed in the HPLC system sampler at 25°C ± 2.5°C. HPLC analysis started 24 hours ± 2 hours after addition of peptides.
Immediately upon addition of the test item solution to the peptide solution, just prior the beginning of the HPLC analysis and at the end ofthe analysis, samples were checked. No precipitate was observed.

Results and discussion

Positive control results:
Depletion in Lysine Peptide % (mean): 51.55
Depletion in Cysteine Peptide % (mean): 75.11

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
other: mean (1-3)
Parameter:
other: Depletion in Lysine Peptide %
Value:
0
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: mean (1-3)
Parameter:
other: Depletion in Cysteine Peptide %
Value:
2.87
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
mean
Parameter:
other: Mean Depletion % (Lysine and Cysteine Peptide)
Value:
1.44
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The following table allows a detennination of reactivity: the 6.38% threshold is used in order to differentiate between non-sensitizers and sensitizers.

 Mean of cysteine and lysine% depletion  Reactivity Class  DPRA Prediction
 0.00 %  <= mean% depletion  <= 6.38 %  No or minimal reactivity  Negative
 6.38 % < mean% depletion  <= 22.62 %  Low reactivity  Positive
 22.62 % < mean% depletion  <= 42.47 % Moderate reactivity   Positive
 42.4 7 % < mean% depletion <= 100 %  High reactivity  Positive

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The sensitivity of the test item is determined by calculating the mean percentage of depletion of Lysine and Cysteine.
The test item, TRIMETHYL PHOSPHATE - 97% shows mean depletion of 0% for Lysine and 2.87 % for Cysteine, i.e. an overall average of 1.44 % reflecting no or minimal reactivity and therefore a negative prediction of DPRA.
The test method DPRA is considered scientifically valid to be used as part of an integrated approach to testing and assessment, to support the identification of the sensitization potential of test item for hazard classification and labeling purposes.