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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008, including most recent amendments
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date certificate 3 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: off-white powder
- Storage condition of test material: At room temperature

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: 20.3 - 23.8 g. Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeled 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 CONDITIONS (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 6 January 2016 to 25 January 2016

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 10, 25, 50% (w/w) Broomdex
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on days 1 and 3, and on day 6. Animals were sacrificed after the final observation.
Based on the results of the range finding test, the highest test item concentration selected for the main study was a 50% concentration.

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 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 and recommendations done by ICCVAM.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with Dimethyl formamide (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.
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. The vehicle was chosen from the vehicles specified in the test guideline.


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 within 1 hour 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.
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 WIl Research Europe is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
50%
Cellular proliferation data / Observations:
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 971, 1191 and 1099 DPM, respectively. The mean DPM/animal value for the vehicle control group was 728 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.3, 1.6 and 1.5, respectively.

Any other information on results incl. tables

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the pre-screen animals. Scaliness was noted for one animal treated at 50% on day 6. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White staining of the dorsal surface of the ears by test item remnants was noted for all animals (days 1-4). The staining did not hamper the scoring of the ears. Based on these results, the highest test item concentration selected for the main study was a 50% concentration.

Other results - main study:

No irritation was observed in any of the animals. Scaliness was noted for all experimental animals on day 6. White staining of the dorsal surface of the ears by test item remnants was noted for all experimental animals (days 1-4). The staining did not hamper the scoring of the ears. 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. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

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, Broomdex was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 50% w/w.
Executive summary:

An LLNA skin sensitisation study was performed with Broomdex according to OECD/EC test guidelines and in compliance with GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 10%, 25% and 50% w/w. No irritation was observed in any of the animals. Scaliness was noted for all experimental animals on Day 6. 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. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 971, 1191 and 1099 DPM, respectively. The mean DPM/animal value for the vehicle control group was 728 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.3, 1.6 and 1.5, respectively. As the SI appeared not to be ≥ 3 when tested up to 50% w/w, Broomdex was considered not to be a skin sensitiser.

Based on these results, Broomdex 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 according to the regulation (EC) No 1272/2008 and has no obligatory labelling requirement for sensitization by skin contact.