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Diss Factsheets

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
Study period:
17 October 2017 - 29 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in chemico skin sensitization tests is the Direct Peptide Reactivity assay (DPRA), which is recommended in international guidelines (e.g. OECD).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
The purpose of this study was to evaluate the reactivity of the test substance towards model synthetic peptides containing either cysteine or lysine, and to categorize the test item in one of four classes of reactivity for supporting the discrimination between skin sensitizers and nonsensitizers

Test material

Constituent 1
Reference substance name:
Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one
EC Number:
915-318-3
Cas Number:
41199-19-3, 17283-81-7 and 31499-72-6
IUPAC Name:
Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light container flushed with nitrogen

In chemico test system

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

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.
Stock solutions of SPCC and SPCL were prepared by dissolving 10 mg of the peptide in 19.96 mL phosphate buffer or 19.31 mL ammonium acetate buffer, respectively. The reference control solutions (0.5 mM) were prepared by diluting the stock solutions with acetonitrile. The samples for the calibration curves for both peptides were prepared at concentrations 0.017, 0.033, 0.067, 0.133, 0.267 and 0.533 mM.
Cinnamic aldehyde at concentration 100 mM in acetonitrile was used as a positive control.
Test item solution was prepared in methanol at concentration 100 mM based on the results of preliminary solubility tests.

Sample incubations:
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in an incubator at 25 ± 2.5 °C. After incubation, the samples were transferred to the autosampler. The incubation time between placement of the samples in the incubator and analysis of the first RCcysB- or RClysB- sample was 24.5 hours. The time between the first RCcysB- or ClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.

Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA at 220 nM. 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)] 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%
Acceptability criteria:
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an 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.

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 in the section "Any other information on materials and methods incl. tables"), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Results and discussion

Positive control results:
The mean Percent SPCC Depletion and mean Percent SPCL Depletion for the positive control cinnamic aldehyde were 74.9% ± 1.8% and 57.3% ± 2.9%, respecitvely. These values were within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%) for SPCC and of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%) for SPCL.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: % SPCC depletion
Value:
1.9
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2
Parameter:
other: % SPCC depletion
Value:
1.2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3
Parameter:
other: % SPCC depletion
Value:
2.4
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1
Parameter:
other: % SPCL depletion
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2
Parameter:
other: % SPCL depletion
Value:
0.7
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3
Parameter:
other: % SPCL depletion
Value:
5.9
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY: the suitability of the HPLC system was demonstrated by measuring the reference control samples for SPCC and SPCL. The mean peptide concentrations of reference control sames were all within the acceptance criteria of 0.50 ± 0.05 mM. The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls was 1.0% for SPCC and 1.9% for SPCL. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 17.81 for SPCC and 16.21 for SPCL. The mean A220/A258 ratio ± 10% range were 16.03-19.59 and 14.59-17.83, respectively. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes, the mean Percent SPCC Depletion and mean Percent SPCL Depletion for the positive control cinnamic aldehyde were 74.9% ± 1.8% and 57.3% ± 2.9%, respecitvely. These values were within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%) for SPCC and of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%) for SPCL.
- Acceptance criteria met for variability between replicate measurements: yes, the standard deviations were 0.6% and 3.2%, which is below the acceptability criteria of 14.9% and 11.6% for SPCC and SPCL depletion, respectively.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
This study is part of a weight of evidence approach on which the classification is based.
Conclusions:
Based on the outcome of a Direct Peptide Reactivity Assay (DPRA) performed according to OECD guideline and GLP principles, Polyambrol is classified as negative (no depletion of synthetic peptides containing either cysteine or lysine) under the experimental conditions described in this report.