Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 - 17 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
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 (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): VC
- Substance type: organic
- Physical state: solid (under storage conditions)
- Test material was warmed at 25°C in a warming bath to produce a liquid before use.
- Analytical purity: >99.9%
- Lot/batch No.: 0801102
- Expiration date of the lot/batch: 31 December 2008
- Stability under storage conditions: stable
- Storage condition of test material: In refrigerator (2-8°C) protected from light and under nitrogen. Avoid contact with air and water.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 19-23 g
- Housing: Individual housing in labeled Macrolon cages
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. Accommodation was as described above except that the animals were group housed in Macrolon cages


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 – 23.5
- Humidity (%): 32 - 74
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Preliminary study: 100, 50, 25, 10, 5, 2.5 and 1%
Main study: 50, 25, 10 and 0%
No. of animals per dose:
5
Details on study design:
- Compound solubility: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. The test substance was heated in a water bath with a maximum temperature of 26°C for a maximum of 4 hours and 13 minutes in order to obtain a liquid before use. Homogeneity was obtained to visually acceptable levels.

RANGE FINDING TESTS:
- Irritation: No irritation was observed in any of the animals examined. The animal at 100% showed hunched posture, piloerection, uncoordinated movements, hypothermia and body weight loss on Day 3. No symptoms of systemic toxicity were noted in the animal at 50%.
Based on the results, the highest test substance concentration selected for the main study was a 50% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- 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 sensitiser, based on the test guideline and recommendations done by ICCVAM.
The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the EC criteria for classification and labeling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published in the Official Journal of the European Communities).
Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3).


TREATMENT PREPARATION AND ADMINISTRATION: by open application on the ears
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.
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.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.

Results and discussion

Positive control results:
The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10 and 25% were 3.3 and 2.1 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Median DPM/animal values are reported due to possible outlier responses for individual animals within groups. Median DPM/animal values for the experimental groups treated with test substance concentrations 10 and 25% were 1689 and 1103 respectively. The median DPM/animal value for the vehicle control group was 518.

Any other information on results incl. tables

On Day 6, two animals at 50% showed hunched posture and piloerection. In addition, marked body weight loss was noted among the animals at 50%. Based on signs of systemic toxicity, the results of the group at 50% could not be used for interpretation.

No skin reactions were observed in any of the control animals and animals at 10 and 25% examined.

All nodes of the control group and groups at 10 and 25% were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted.

Body weights and body weight gain of experimental animals at 10 and 25% remained in the same range as controls over the study period.

No mortality occurred and no symptoms of systemic toxicity were observed in the control animals and animals at 10 and 25% of the main study.

The results indicate that the test substance could elicit a SI >= 3.

The data showed an inverse dose-response and no reliable EC3 value could be calculated. The inverse dose response might be due to underlying systemic toxicity, since animals at 50% showed signs of systemic toxicity. It was possible to strengthen the outcome of the study by adding lower concentrations. Since the SI value at 10% exceeded 3 and since extension of the study would not alter the classification, this was considered not appropriate for ethical reasons.

Applicant's summary and conclusion

Interpretation of results:
other: skin sensitizer
Conclusions:
Based on the results of this study, it can be concluded that:
- according to the recommendations made in the test guidelines, VC would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2004), VC should be classified as skin sensitizer (Category 1).
- according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), VC should be labeled as: may cause sensitization by skin contact (R 43).

Executive summary:

Assessment for Contact Hypersensitivity to VC in the Mouse (Local Lymph Node Assay) was performed according to OECD 429. In the main study, three groups of five experimental animals were treated with test substance concentrations of 10%, 25% or 50% on three consecutive days, by open application on the ears. The SI values calculated for the substance concentrations 10 and 25% were 3.3 and 2.1 respectively, indicating that the test substance could elicit a SI>= 3. Based on these results, VC should be regarded as skin sensitizer.