Disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2016 - June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was conducted before guidance on in vitro testing strategy was implemented.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

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)

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008, including most recent amendments

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 03 November 2015

Type of study:

mouse local lymph node assay (LLNA)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: yellow/brown powder
- Storage conditions: in refrigerator (2-8°C)

Specific details on test material used for the study:

pH (1% in water, indicative range): 6.2 – 6.4 (determined by Charles River Den Bosch)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 18.5 - 24.3 g
- Housing: Animals were group housed in labeled Makrolon cages. Shelters were supplied as cage-enrichment.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 10
- Photoperiod (hrs dark / hrs light): 12/12

There were no deviations in environmental conditions that affected the integrity of the study.

IN-LIFE DATES: From: 5 April to 3 May 2016

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 2, 10, 20% w/w 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonic acid

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 10% and 20% concentration. The highest concentration was the highest concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on days 1 and 3, and on day 6. Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.
Rationale for vehicle: The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at WIL Research Europe.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on days 1-6 (on days 1 - 3 immediately after dosing) according to a numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy for gross macroscopic examination was performed according to study plan.

- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer, based on the test guideline.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Laboratories Den Bosch is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

< 3

Cellular proliferation data / Observations:

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 10 and 20% were 1185, 1314 and 1575 DPM, respectively. The mean DPM/animal value for the vehicle control group was 880 DPM. The SI values calculated for the test item concentrations 2, 10 and 20% were 1.3, 1.5 and 1.8, respectively.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Laboratories Den Bosch is an appropriate model for testing for contact hypersensitivity.

Any other information on results incl. tables

Results Pre-screen test:

No signs of systemic toxicity were observed in any of the pre-screen animals. Very slight erythema was noted for one animal treated at 10% and both animals treated at 20% on day 3 only. Scaliness was noted for the animals treated at 20% on day 6.Yellow staining of test item remnants on the dorsal surface of the ears did not hamper scoring for erythema. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test item concentration selected for the main study was a 20% concentration.

Other results - main study:

No irritation was observed in any of the animals.The scaliness as shown by the experimental animals between Days 4 and 6 and scabs as shown by two animals treated at 20% on Day 6 was considered not to have a toxicologically significant effect on the activity of the nodes. Yellow staining of test item remnants on the dorsal surface of the ears of the experimental animals did not hamper scoring for erythema.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

 

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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonic acid was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to and including 20% w/w.

Executive summary:

An LLNA skin sensitisation study was performed with 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonic acid according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 2%, 10% and 20% (w/w). No irritation of the ears was observed in any of the animals. The scaliness as shown by the experimental animals between days 4 and 6 and scabs as shown by two animals treated at 20% on day 6 was considered not to have a toxicologically significant effect on the activity of the nodes. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. 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. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Laboratories Den Bosch is an appropriate model for testing for contact hypersensitivity. Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 10 and 20% were 1185, 1314 and 1575 DPM, respectively. The mean DPM/animal value for the vehicle control group was 880 DPM. The SI values calculated for the test item concentrations 2, 10 and 20% were 1.3, 1.5 and 1.8, respectively. As the SI appeared not to be ≥ 3 when tested up to and including 20% w/w, 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonic acid was considered not to be a skin sensitiser.

Based on these results, 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonic acid is not regarded as a skin sensitizer and is not classified for sensitization by skin contact.