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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation
Remarks:
other: in vitro DPRA
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 February 2014 to 19 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to draft OECD test guideline.
Qualifier:
according to guideline
Guideline:
other: Draft OECD 13Nov2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: DPRA
Positive control results:
The positive control substance induced a mean depletion rate of 60.47%.
Key result
Run / experiment:
mean
Parameter:
other: Cysteine peptide depletion
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
mean
Parameter:
other: Lysine peptide depletion
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

Acceptance criteria were as per the draft OECD guideline.

Classification criteria for direct peptide reactivity were as per the draft OECD guideline.

Results for the test item and the positive control are presented in attached Tables 1 and 2, respectively. The peak areas for calibration curve samples are presented in attached Tables 3.1 and 3.2, the peptide concentrations and peak areas in reference control samples are presented in attached Tables 4.1 and 4.2. Representative chromatograms of the co-elution, reference control C and test item samples are presented for each peptide in attached Figures 1 to 6. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples (Figures 1 and 4) indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation. For both cysteine and lysine peptides, the mean depletion values were set to 0% since the individual percent depletion values were negative. The mean of the percent cysteine and percentage lysine depletions was therefore equal to 0%. Accordingly, the test item was considered to have no/minimal peptide reactivity.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other:
Conclusions:
Under the experimental conditions of this study, the test item, has 0% peptide reactivity.
Executive summary:

Introduction

The objective of the Direct Peptide Reactivity Assay (DPRA) was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides for skin sensitization assessment.

Methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the amount of peptide remaining in the sample relative to the average amount measured in the relevant reference control.

Results

Solubility assay

The test item was found soluble at 50 mg/mL in water obtained by reverse osmosis. Therefore, water obtained by reverse osmosis was selected as vehicle.

Direct Peptide Reactivity Assay

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated. For both cysteine and lysine peptides, the mean depletion values were set to 0% since the individual percent depletion values were negative. The mean of the percent cysteine and percent lysine depletions was therefore 0%. Accordingly, the test item was considered to have no/minimal peptide reactivity.

Conclusion

Under the experimental conditions of this study, the test item, has 0% peptide reactivity

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Key In vitro Direct Peptide Reactivity Assay (DPRA)

Introduction

The objective of the Direct Peptide Reactivity Assay (DPRA) was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides for skin sensitization assessment.

Methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm. Peptide reactivity was reported as percent depletion based on the amount of peptide remaining in the sample relative to the average amount measured in the relevant reference control.

Results

Solubility assay

The test item was found soluble at 50 mg/mL in water obtained by reverse osmosis. Therefore, water obtained by reverse osmosis was selected as vehicle.

Direct Peptide Reactivity Assay

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid. Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated. For both cysteine and lysine peptides, the mean depletion values were set to 0% since the individual percent depletion values were negative. The mean of the percent cysteine and percent lysine depletions was therefore 0%. Accordingly, the test item was considered to have no/minimal peptide reactivity.

Conclusion

Under the experimental conditions of this study, the test item, has 0% peptide reactivity

Disregarded In vivo LLNA Study

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

In order to study the possible contact allergenic potential of the test substance, three groups of five female mice each were treated daily with the test item at concentrations of 25%, 50% and 75% (w/v) in acetonerolive oil (4:1 v/v) by topical application to the dorsum of each ear lobe for three consecutive days. A control group of five female mice was treated with the vehicle acetone:olive oil (4:1 v/v) only. A concurrent positive control group of five mice was treated with 25% (w/v) alpha-hexylcinnamaldehyde in acetone:olive oil (4:1 v/v). Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine ( 3 H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised and pooled per animal. For each animal, single cell suspensions of lymph node cells were prepared and subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured in a p-scintillation counter.

Results

All treated animals survived the scheduled study period. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period. The results obtained [Stimulation Index (S.I.)] are reported in the following table. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.:

Treatment Group

Concentration

Stimulation Index

Result

Test Item

25% w/w in
acetone/olive oil (4:1 v/v)

2.6

Negative

10% w/w in
acetone/olive oil (4:1 v/v)

7.2

 Positive

50% w/w in
acetone/olive oil (4:1 v/v)

6.6

 Positive

Positive
Control Item

75% v/v in

acetone/olive oil (4:1 v/v)

8.9

Positive

The concentration of test item expected to cause a 3 fold increase in 3 HTdR incorporation (EC 3 value) was calculated to be 27.2%.

Conclusion

In conclusion, the test item was found to produce an EC3 value of 27.2% (w/v) and was considered to be a potential skin sensitizer under the conditions of the test.


Migrated from Short description of key information:
Although a GLP study, classified as K1, using the OECD 429 mouse lymph node assay (LLNA) performed on the test substance after formulation in acetone/olive oil (4:1, v/v) at 25, 50 and 75% w/w. indicated that the substance was potentially sensitising, a confirmation Direct Peptide Reactivity Assay (DPRA) showed that the substance has a 0% peptide reactivity. The substance is therefore not a skin sensitiser.

Justification for selection of skin sensitisation endpoint:
A GLP study classified as K1, using the draft OECD Direct Peptide Reactivity Assay (DRPA) test guideline provided a negative (non peptide reactive and therefore unlikely to be a skin sensitizer) result
A GLP study, classified as K3, using the OECD 429 mouse lymph node assay (LLNA) provided a positive (sensitizing) conclusion. However, the study was disregarded because it is not reliable for this class of chemical.

Justification for classification or non-classification

Although a GLP study, classified as K1, using the OECD 429 mouse lymph node assay (LLNA) performed on the test substance after formulation in acetone/olive oil (4:1, v/v) at 25, 50 and 75% w/w indicated that the substance was potentially sensitising, a confirmation Direct Peptide Reactivity Assay (DPRA) showed that the substance has a 0% peptide reactivity. The substance is therefore not a skin sensitiser.