4-allylveratrole

Endpoint summary

Description of key information

Skin sensitisation by in chemico test method, Direct Peptide Reactivity Assay (DPRA) was carried out using the nucleophile containing synthetic cysteine and lysine peptides to evaluate skin sensitisation potential of the test item 4 -allylveratrole (Methyl Eugenol). The mean percent depletion was 4.90% for cysteine peptide and 1.09% for lysine peptide. The mean of cysteine and lysine peptide depletion was 3.00% which shows that the test item has no or minimal activity according to cysteine 1:10/lysine 1:50 prediction model. Under the same conditions positive control cinnamaldehyde showed 71.03% and 35.59% mean cysteine and lysine peptide depletion confirming the sensitivity of the assay. Under the test conditions, test item, 4 -allylveratrole, Methyl Eugenol was concluded as negative in the skin sensitisation assay by direct peptide reactivity assay (DPRA).

Skin sensitisation potency of 4 -allylveratrole, Methyl Eugenol was studied in guinea pigs in a study equivalent to OECD guideline 406 (Itoh 1982). Induction exposure was performed with 0.2 -10% of the test item for 48 hours. Following challenge exposure duration was 48 hours and concentrations used 0.1% and 1 %. Skin was evaluated after 1, 24, 48 hours and one week after challenge. No animals were sensitised by 4 -allylveratrole, Methyl Eugenol under test conditions.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Oct 2017-28 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The correlation of protein rectivity with skin sensitisation potential is well established. Haptenation i.e. covalent binding of low molecluar weight substances (Haptens) to proteins present in skin is considered a prominent mechanism through which chemicals or their metabolites become antigenic. Therefore, information inferred from the DRPA assay is relevant for assessment for skin sensitisation potential of substances.

Details on the study design:

Solutions of test item and positive control prepared at 100 mM concentration were used in the sample preparation for analysis. Test solution, positive control and reference control was added to cysteine and lysine peptides and incubated in dark at 25°C for 24±2 hours. The test samples were then loaded in HPLC auto sampler to measure the peptide depletion. The test samples were then analysed by reversed-phase HPLC coupled with UV detector for detection of peptides at 220nm. The HPLC column Zorbax SBC18 (2.1mm X100mm X 3.5 micron) was equilibrated at 30°C with 50% phase A (0.1% (v/v) trifluoroacetic acid in water) and 50% phase B (0.085% (v/v) trifluoroacetic acid in acetonitrile). Analysis was carried out at a flow rate of 0.35 mL/min, with sample injection volume range of 10 μL. A linear gradient from 10% to 25% Acetonitrile over 10 minutes, was used for separation, followed by a rapid increase to 90% acetonitrile to remove other materials. Cysteine and lysine peptide Percent Depletion Values were then calculated from peptide peak areas obtained from the HPLC analysis.

Positive control results:

Cinnamaldehyde showed 71.03% mean cysteine depletion and 35.59% mean lysine peptide depletion.

Key result

Parameter:

other: % of cysteine peptide depletion

Value:

4.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other: % of lysine peptide depletion

Value:

1.09

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1. Reactivity class classification of test item and positive control as per cysteine 1:10 lysine 1:50 prediction model.

Sample ID	Name of the chemical	Mean % of cysteine peptide depletion	Mean % of lysine peptide depletion	Mean peptide % peptide depletion	Reactivity class	DPRA prediction
Y02 -01	4 -allylveratrole, Methyl Eugenol	4,90	1,09	3,00	No or minimal acticity	Negative
CA	Cinnamic aldehyde	71,03	35,59	53,31	High reactivity	Positive

Interpretation of results:

GHS criteria not met

Conclusions:

The mean percent depletion was 4.90% for cysteine peptide and 1.09% for lysine peptide. The mean of cysteine and lysine peptide depletion was 3.00% which shows that the test item has no or minimal activity according to cysteine 1:10/lysine 1:50 prediction model. Hence it was concluded that the test item 4-allyveratrole, Methyl Eugenol is negative in skin sensitisation test by DPRA.

Executive summary:

Skin sensitisation by in chemico test method, Direct Peptide Reactivity Assay (DPRA) was carried out using the nucleophile containing synthetic cysteine and lysine peptides to evaluate skin sensitisation potential of the test item 4 -allylveratrole (Methyl Eugenol). The mean percent depletion was 4.90% for cysteine peptide and 1.09% for lysine peptide. The mean of cysteine and lysine peptide depletion was 3.00% which shows that the test item has no or minimal activity according to cysteine 1:10/lysine 1:50 prediction model. Under the same conditions positive control cinnamaldehyde showed 71.03% and 35.59% mean cysteine and lysine peptide depletion confirming the sensitivity of the assay. Under the test conditions, test item, 4 -allylveratrole, Methyl Eugenol was concluded as negative in the skin sensitisation assay by direct peptide reactivity assay (DPRA).

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Published data (Itoh M 1981).

Species:

guinea pig

Strain:

Hartley

Sex:

female

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

0.2, 0.5, 1, 2, 5, 10%

Day(s)/duration:

2 days

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

0.1%

Day(s)/duration:

2 days

Adequacy of challenge:

not specified

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

1%

Day(s)/duration:

2 days

Adequacy of challenge:

not specified

No. of animals per dose:

10

Details on study design:

RANGE FINDING TESTS: skin irritation results were used, published in the same paper (Itoh, 1982)

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 6
- Exposure period: 48 hours per application
- Test groups: 10 per compound tested (severeal different compounds were tested in this publication)
- Site: nape
- Frequency of applications: 3 times a week
- Duration: 2 weeks
- Concentrations: 50 mg (methyl eugenol in white petrolatum)

B. CHALLENGE EXPOSURE
- Day(s) of challenge: two weeks after the end of the induction test
- Exposure period: 48 hours
- Site: flank
- Concentrations: 0.1% and 1%
- Evaluation (hr after challenge): 1, 24, 48 hours and one week

Positive control substance(s):

not required

Remarks:

14 phenolic compounds were studied for sensitisation potential

Key result

Reading:

other: mean

Hours after challenge:

72

Group:

test chemical

Dose level:

0.1% and 1%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading

Interpretation of results:

other: not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

No animals were sensitised by methyl eugenol under these test conditions. Therefore the test substance is not classified as skin sensitising.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The outcome of in chemico test method, Direct Peptide Reactivity Assay (DPRA) was negative for the test item. Moreover, in an in vivo study in guinea pigs no animals were sensitised by the test item using protocol similar to OECD 406 guideline. Therefore, 4 -allylveratrole, methyl eugenol is classified as not skin sensitising.