Reaction products of diazotized mixture of aniline, toluidines and xylidines coupled with mixture of aniline, toluidines and xylidines, subsequently diazotized then coupled with 2-naphthol

Skin sensitisation

Administrative data

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diazotized mixture of aniline, toluidines and xylidines coupled with mixture of aniline, toluidines and xylidines, subsequently diazotized then coupled with 2-naphthol

EC Number:

947-944-8

Cas Number:

70879-65-1

Molecular formula:

C23H17N4O

IUPAC Name:

Reaction products of diazotized mixture of aniline, toluidines and xylidines coupled with mixture of aniline, toluidines and xylidines, subsequently diazotized then coupled with 2-naphthol

Specific details on test material used for the study:

For the purpose of the study, the test item was used freshly prepared in Methyl ethyl ketone (MEK). This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given in the procedure section. The vehicle determination record is included in Appendix 3.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Female CBA/J (CBA/JRj) strain mice (SPF caw) were nulliparous and non-pregnant. After an acclimatisation period of at least five days under stabling and nutritional conditions identical to those of the test, the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the main study the animals were 8 or 9 weeks old. The animals were weighed at the beginning and at the end of the study.
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. The temperature and relative humidity were controlled to remain within target ranges of 19°C to 25°C and 30% to 70%, respectively. The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness. The drinking water and food were supplied ad libitum.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

5, 10 and 20% in the main study

No. of animals per dose:

4 females/dose group

Details on study design:

Groups of four mice were treated with the test item diluted at 20%, 10% and 5% in methyl ethyl ketone. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone (methyl ethyl ketone) in the same manner.
0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route on day 5. On day 6 (end of the test), the animals were euthanized with sodium pentobarbital (Dolethal®).

Positive control substance(s):

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

Results and discussion

Positive control results:

Based on the EC1.6 value (18.51%), the substance alpha-Hexylcinnamaldehyde in accordance with the Regulation (EC) No. 1272/2008 has to be classified in category 1 “Skin sensitisation”. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.36

Test group / Remarks:

5% dose group

Results 2

Key result

Parameter:

SI

Value:

ca. 1.53

Test group / Remarks:

10% dose group

Results 3

Key result

Parameter:

SI

Value:

ca. 1.37

Test group / Remarks:

20% dose group

Any other information on results incl. tables

Mortality: No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

Weight evolution: Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Estimation of the proliferative response of lymph node cells: The Stimulation Index (SI) calculated by individual approach was 1.36, 1.53 and 1.37 for the treated groups at 5%, 10% and 20%, respectively. Therefore, the EC1.6 cannot be determined due to the absence of SI value higher than 1.6.

Local irritation: Red coloration not preventing erythema quotation was noted in animals treated at 5%, 10% and 20% between days 2 and 6. A slight increase in ear thickness (+1.5%, +5.1%, +5.0%) was noted in animals treated at 5%, 10% and 20%, respectively. A slight increase in ear weight (+2.3%, +8.0%, +7.0%) was noted in animals treated at 5%, 10% and 20%, respectively. Therefore, the test item has to be considered as not excessively irritant at these concentrations.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained, under these experimental conditions, enable to conclude that the test item SOLVENT RED 24 MIX does not have to be classified as a skin sensitizer, in accordance with the Regulation EC No. No. 1272/2008 on classification, labelling and packaging of substances and mixtures. No signal word or hazard statement is required.