N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

Endpoint summary

Description of key information

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 40%, Disperse Brown 27 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 40%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, Disperse Brown 27 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

The batch of Disperse Brown 27 (4852-1501) tested was a brown powder with a purity of 99.7 %

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

20 females (nulliparous and non-pregnant), five females per group (main study only) were used for the study.
Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Subjects were identified by tail mark with a marker pen.
At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.
The reliability of test results with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures were evaluated during the study. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study. An extensive data base is available with reliability checks performed at half year intervals during at least the past 9 years showing reproducible and consistent positive results.

Vehicle:

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

Concentration:

4 different concentration groups, 0, 10, 25 and 40% w/w of test item.

No. of animals per dose:

The pre-screen test consisted of 2 dose groups made up of 2 animals, four animals in total.
For the main study there were 5 animals per dose, 20 animals in total

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Key result

Parameter:

SI

Value:

ca. 1.5

Test group / Remarks:

Test item conc 10% w/w

Key result

Parameter:

SI

Value:

ca. 1.9

Test group / Remarks:

Test item conc 25% w/w

Key result

Parameter:

SI

Value:

ca. 2

Test group / Remarks:

Test item conc 40% w/w

During the pre-screen test very slight erythema was noted for both animals treated at 25% between Days 3 and 6. Very slight to well-defined erythema was noted for both animals at 40% between Days 3 and 6. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Brown test item remnants were present on the dorsal surface of the ears of all pre-screen animals throughout the observation period, which did not hamper scoring of the skin reactions. No signs of systemic toxicity were observed in any of the pre-screen animals. Based on these results, the highest test item concentration selected for the main study was a 40% concentration.

During the main study no erythema was observed in any of the animals. Brown test item remnants were present on the dorsal surface of the ears of all test item treated animals throughout the observation period, which did not hamper scoring of the skin reactions.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Yellow discoloration of the tissue surrounding the nodes was noted for all animals treated at 25 and 40%. No further macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 40% were 588, 733 and 788 DPM, respectively. The mean DPM/animal value for the vehicle control group was 393 DPM. The SI values calculated for test item concentrations 10, 25 and 40% were 1.5, 1.9 and 2.0, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 40%, Disperse Brown 27 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 40%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Based on these results, Disperse Brown 27 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

Test item concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 40% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Yellow discoloration of the tissue surrounding the nodes was noted for all animals treated at 25 and 40%. No further macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 40% were 588, 733 and 788 DPM, respectively. The mean DPM/animal value for the vehicle control group was 393 DPM. The SI values calculated for the test item concentrations 10, 25 and 40% were 1.5, 1.9 and 2.0, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 40%, Disperse Brown 27 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 40%.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 40%, Disperse Brown 27 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 40%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, Disperse Brown 27 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).