Registration Dossier

Administrative data

Description of key information

Female CBA/J mice (5/dose) were topically treated with 0, 25, 50 and 100% of the test substance in methyl ethyl ketone in an LLNA skin sensitisation study performed according to OECD 429.

No irritation of the ears was observed in any of the animals examined. No test substance related 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 the majority of the experimental animals remained in the same range as controls over the study period. The majority of auricular lymph nodes examined were enlarged and the largest auricular lymph nodes were found in the higher dose groups.

The SI values calculated for the substance concentrations 25, 50 and 100% were 2.2, 8.3 and 8.4, 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 28.3% was calculated. Therefore, the test substance is considered a moderate skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: EXP1200078
Description: Dark brown viscous liquid
Batch: E00275-350
Expiration: 31 December 2013
Species:
mouse
Strain:
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. 9 weeks old)
- Weight at study initiation: 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
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 - 22 April 2013
Vehicle:
methyl ethyl ketone
Concentration:
0, 25, 50 and 100%
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:
Two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids).

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.

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. At regular intervals the sensitivity of the test system was checked by treating five animals with the positive control substance, hexylcinnamaldehyde.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. In order to obtain homogeneity to visually acceptable levels and to facilitate dosing, the test substance (formulations) and vehicle were kept at a maximum of 40 ºC.

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.

Erythema and eschar formation:
No erythema ..............................................................................……………………………………….. ......... 0
Very slight erythema (barely perceptible) ....................................................……………………………… .... 1
Well-defined erythema ...................................................................…………………………………… .......... 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) .........……… ... 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema .........……… ......... 4

Necropsy: No necropsy was performed on the animal that died on day 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Linear interpolation.
Positive control results:
The six-month reliability check with 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'.
Key result
Parameter:
EC3
Value:
28.3
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100% were 2.2, 8.3 and 8.4, respectively. The EC3 value was calculated to be 28.3%.
Parameter:
SI
Value:
1
Variability:
+/-0.1
Test group / Remarks:
Control group
Parameter:
SI
Value:
2.2
Variability:
+/-0.6
Test group / Remarks:
25% concentration applied
Parameter:
SI
Value:
8.3
Variability:
+/-2.2
Test group / Remarks:
50% concentration applied
Parameter:
SI
Value:
8.4
Variability:
+/-1.7
Test group / Remarks:
100% material (neat/undiluted)

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. Brown staining of the dorsal surface of the ears by test substance remnants was noted for animals 1 and 2 on Days 2 and 3 and for animals 3 and 4 between Days 1 and 4. The staining did not hamper the scoring of the ears. 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 100% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined. Brown staining of the dorsal surface of the ears by test substance remnants was noted for group 3 (Days 2 and 3) and for group 4 (between Days 1 and 3 and for animal 17 Day 4 right ear only). The staining did not hamper the scoring of the ears.

 

No test substance related 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 the majority of the experimental animals remained in the same range as controls over the study period. The body weight loss noted across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent. One low dose animal showed a body weight loss that was just outside the study range. This animal

died prior to the injection of the 3H-methyl thymidine. It was considered that the death of this animal might have been triggered by the experimental procedures on Day 6.

The majority of auricular lymph nodes examined were enlarged and the largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD 429, the substance was considered to be a skin sensitiser, as the SI appeared to be = 3 when tested up to 100%.
Executive summary:

Female CBA/J mice (5/dose) were topically treated with 0, 25, 50 and 100% of the test substance in methyl ethyl ketone in an LLNA skin sensitisation study performed according to OECD 429.

No irritation of the ears was observed in any of the animals examined. No test substance related 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 the majority of the experimental animals remained in the same range as controls over the study period. The majority of auricular lymph nodes examined were enlarged and the largest auricular lymph nodes were found in the higher dose groups.

The SI values calculated for the substance concentrations 25, 50 and 100% were 2.2, 8.3 and 8.4, 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 28.3% was calculated. Therefore, the test substance is considered a moderate skin sensitizer.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study waived due to provisions of other regulation
Justification for data waiving:
other:
Executive summary:

Study does not to be conducted because an mLLNA conducted as a requirement by a non-EU, non-European entity.  Furthermore, a valid mLLNA study was completed on13 June 2013.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance to Regulation (EC) No 1272/2008 the registration substance is determined to be a Cat 1B skin sensitizer as the EC3 value is >2%.