4-tert-Butoxystyrene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

April 2002

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-(tert-butoxy)-4-ethenylbenzene

EC Number:

619-127-3

Cas Number:

95418-58-9

Molecular formula:

C12H16O

IUPAC Name:

1-(tert-butoxy)-4-ethenylbenzene

Test material form:

liquid

Details on test material:

Clear and colourless

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: young adult animals (approx. 10 weeks old) were selected.
- Weight at study initiation: body weight variation was within +/- 20% of the sex mean.
- Identification: tail mark with marker pen.
- Housing: Individual housing in labeled Macrolon cages containing sterilized sawdust as bedding material. Paper was supplied as cage-enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: the acclimation 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.
- Indication of any skin lesions: a health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25%-50%-100%

No. of animals per dose:

5 females per dose group

Positive control substance(s):

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

Statistics:

[³H]TdR incorporation was calculated as mean DPM ± SEM, and stimulation indices of lymph node cell proliferation between substance-treated and vehicle-treated groups were calculated as SI ± SEM.

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)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle-treated group

Results 2

Parameter:

SI

Value:

26

Variability:

± 9.9 (SEM)

Test group / Remarks:

100% test substance concentration group

Results 3

Parameter:

SI

Value:

26.5

Variability:

± 8.9 (SEM)

Test group / Remarks:

50% test substance concentration group

Results 4

Key result

Parameter:

SI

Value:

11

Variability:

± 3.6 (SEM)

Test group / Remarks:

25% test substance concentration group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
- Macroscopy of the auricular lymph nodes and surrounding area: The largest nodes were found in the highest dose group. No macroscopic abnormalities of the surrounding area were noted.

DETAILS ON STIMULATION INDEX CALCULATION
The results indicate that the test substance could elicit an SI ≥ 3.

EC3 CALCULATION
No reliable EC value could be calculated.

CLINICAL OBSERVATIONS
- Skin reactions/Irritation: Slight erythema was observed in two animals at 25%, and in all animals at 50% and 100%. No oedema was observed in any of the animals examined. The irritation of the ears as shown by the animals was considered not to have a toxicologically significant effect on the activity of the nodes.
- Toxicity/Mortality: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The SI values calculated for the substance concentrations 25, 50 and 100% were 11.0, 26.5 and 26.0 respectively. These results indicate that the test substance could elicit an SI ≥ 3. No reliable EC value could be calculated. Based on these results, butoxystyrene should be classified as skin sensitizer (Cat. 1) according to GHS criteria.

Executive summary:

A Local Lymph Node Assay (LLNA) was performed to assess contact hypersensitivity to butoxystyrene in mice. Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three groups of five experimental animals were treated with test substance concentrations of 25%, 50% or 100% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil). 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.

Slight erythema was observed in two animals at 25% and in all animals at 50% and 100%. The irritation of the ears as shown by the animals was considered not to have a toxicologically significant effect on the activity of the nodes. The largest nodes were found in the highest dose group. No macroscopic abnormalities of the surrounding area were noted.

The SI values calculated for the substance concentrations 25, 50 and 100% were 11.0, 26.5 and 26.0 respectively. These results indicate that the test substance could elicit an SI ≥ 3. No reliable EC value could be calculated.

Based on these results, butoxystyrene should be classified as skin sensitizer (Category 1) according to GHS criteria.