Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Remarks:

Study was performed for other regulatory jurisdictions and the results incorporated in this registration dossier

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:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts

EC Number:

812-037-7

Cas Number:

1793011-72-9

Molecular formula:

Unknown for all components

IUPAC Name:

Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Inbred, SPF-Quality
- Age at study initiation: Young adult animals (approximately 10 weeks old) were selected.
- Weight at study initiation: 18.2 to 23.5 g
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized sawdust as bedding material.
- Diet (e.g. ad libitum): Pelleted rodent diet fed ab libitum except during designated procedures.
- Water (e.g. ad libitum): Municipal tap-water was freely available to each animal via water bottles.
- Acclimation period: At least 5 days
- Indication of any skin lesions: Animals in poor health or at extremes of body weight range were not assigned to the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Study design: in vivo (LLNA)

Vehicle:

other: Ethanol/Elix water 7:3

Concentration:

Pre-screen test: 25 and 40% concentration
Main test: 10, 25 and 40% concentration

No. of animals per dose:

5 females

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Not applicable as appropriate vehicle used
- Irritation: Assessed and none observe d
- Systemic toxicity: Assessed and none observed
- Ear thickness measurements: Assessed and no effects noted
- Erythema scores: No signs of irritation were noted.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were stirred until and during dosing.

Positive control substance(s):

other: Alpha-hexylcinnamaldehyde in Ethanol/Elix water (7:3)

Statistics:

Not conducted (no EC3 value could be calculated)

Results and discussion

Positive control results:

The positive control group added to the study showed that the vehicle is suitable for eliciting a SI>3 in this batch of animals and with the procedures used for this study. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the test lab is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.7

Variability:

Standard error of the mean +/- 0.4%

Test group / Remarks:

10%

Results 2

Parameter:

SI

Value:

2

Variability:

Standard error of the mean +/- 0.9%

Test group / Remarks:

25%

Results 3

Parameter:

SI

Value:

2.5

Variability:

Standard error of the mean +/- 0.4

Test group / Remarks:

40%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION : The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

EC3 CALCULATION : Could not be calculated as the SI was below 3.

CLINICAL OBSERVATIONS: One animal treated at 40% was found dead right after injection with thymidine which was most likely due to a technical error during thymidine injection and was considered not to be test item related. Sufficient data was available for analysis. No further 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 and positive control animals remained in the same range as vehicle control animals over the study period. Black discoloration of the faeces was noted for all test item treated animals between Days 2 and 5, this was considered to be due to the staining properties of the test item and was considered not indicative of toxicity.

BODY WEIGHTS: No effects noted

Any other information on results incl. tables

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 40% were 360, 427 and 530 DPM, respectively. The mean DPM/animal value for the vehicle control group was 213 DPM and a mean DPM/animal value of 5121 DPM was obtained from the positive control group. The SI values calculated for the test item concentrations 10, 25 and 40% were 1.7, 2.0 and 2.5, respectively. The SI value calculated for the positive control group was 24.0.

Applicant's summary and conclusion

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%, the test substance 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%.