N-bromosuccinimide

Endpoint summary

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

Study period:

23 April - 26 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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)

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: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeld makrolon cages.
- 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 controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 23 April - 26 May 2014

Vehicle:

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

Concentration:

0, 2.5, 5, 10%

No. of animals per dose:

5

Details on study design:

The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to start of this study. All available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (20011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on Classification, Labelling and Packaging of substances and mixtures.

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 substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.
- Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.
- 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 the following 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 protocol.

Positive control substance(s):

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

Statistics:

Not performed.

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 2.5, 5 and 10% were 1.5, 4.1 and 7.8 respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 2.5, 5 and 10% were 1179, 3271 and 6195 DPM respectively. The mean DPM/animal value for the vehicle control group was 794 DPM.

Results Pre-screen test:

Very slight erythema was shown by the animals at 25% (Days 4-6) and 50% (Days 3-6). Scabs were shown by these animals between Days 4 and 6. At the 5% and 10% test substance concentrations no irritation was observed. White test substance remnants were present on the dorsal surface of the ears of both animals at 25% (Days 1-3) and 50% (Days 1-2) and on one ear of one animal at 10% (Day 2) which did not hamper scoring of the skin reactions. No signs of systemic toxicity were observed in any of the animals examined. 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 substance concentration selected for the main study was a 10% concentration.

Other results - main study:

- Skin reactions / Irritation: No irritation of the ears was observed in any of the animals examined.

- Systemic toxicity/Body weights: 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.

- Macroscopy of the auricular lymph nodes and surrounding area:The auricular lymph nodes of the vehicle control animals and animals treated at 2.5% were considered normal in size. The nodes of the animals at 5% and 10% were considered enlarge. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The SI values calculated for the substance concentrations 2.5, 5 and 10% were 1.5, 4.1 and 7.8 respectively. These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 3.9% was calculated.

Executive summary:

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 2.5, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with 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.

No irritation of the ears was observed in any of the animals examined.

The auricular lymph nodes of the vehicle control animals and animals treated at 2.5% were considered normal in size. The nodes of the animals at 5% and 10% were considered enlarged.

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

Mean DPM/animal values for the experimental groups treated with test substance concentrations 2.5, 5 and 10% were 1179, 3271 and 6195 DPM respectively. The mean DPM/animal value for the vehicle control group was 794 DPM. The SI values calculated for the substance concentrations 2.5, 5 and 10% were 1.5, 4.1 and 7.8 respectively.

These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 3.9% was calculated.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Justification for selection of skin sensitisation endpoint:
The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 3.9% was
calculated.

Respiratory sensitisation

Endpoint conclusion

Additional information:

Migrated from Short description of key information:
Assessment of Contact Hypersensitivity to N-Bromo-succinimide in the Mouse (Local Lymph Node Assay).
In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 2.5, 5 or 10% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with 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.

No irritation of the ears was observed in any of the animals examined.
The auricular lymph nodes of the vehicle control animals and animals treated at 2.5% were considered normal in size. The nodes of the animals at 5% and 10% were considered enlarged.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Mean DPM/animal values for the experimental groups treated with test substance concentrations 2.5, 5 and 10% were 1179, 3271 and 6195 DPM respectively. The mean DPM/animal value for the vehicle control group was 794 DPM. The SI values calculated for the substance concentrations 2.5, 5 and 10% were 1.5, 4.1 and 7.8 respectively.
These results indicate that the test substance could elicit an SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 3.9% was calculated.

Justification for classification or non-classification

The data showed a dose-response and an EC3 value of 3.9% was calculated.

Based on these results:

- according to the recommendations made in the test guidelines (including all amendments), NBromo- succinimide would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), N-Bromo-succinimide should be classified as skin sensitizer (Category 1B).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), N-Bromo-succinimide should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.