Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 August 2009 - 14 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to EC and/or OECD guidelines and in compliance with GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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): Terate® 091 Residue
- Substance type: Dark brown solid with soft lumps
- Physical state: Solid
- Analytical purity: 100%
- Composition of test material, percentage of components: by-product from the manufacture of 1,4-Benzenedicarboxylic acid, dimethyl ester
- Lot/batch No.: NB8322-091
- Expiration date of the lot/batch: 14 July 2010
- Stability under test conditions: Not indicated
- Storage condition of test material: At room temperature in the dark

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: approximately 10 weeks old
- Weight at study initiation: 20-24 gram
- Housing: Individual housing in labeled Macrolon cages containing sterilized sawdust as bedding material . Paper was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days groupo housed under laboratory conditions.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 - 23.5ºC
- Humidity (%): 42 - 74%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 26 August 2009 To: 14 September 2009

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 - 10 - 25 - 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Homogeneity was obtained to visually acceptable levels.
- Irritation: slight erythema of one ear of the animal treated at 50% test substance 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. 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, based on the test guideline and recommendations done by ICCVAM (Reference 1).



TREATMENT PREPARATION AND ADMINISTRATION:
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. Homogeneity was obtained to visually acceptable levels.
Three groups of five animals were treated with test substance concentration of 10, 25 or 50% on three consecutive days. The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone (Acetone/Olive oil (4:1 v/v)) was administered.
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.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistical analysis was performed.

Results and discussion

Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 2.0, 2.2 and 4.2 respectively. An EC3 value of 16.0 % was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 13.1, 15.6, 14.1, 13.8 and 13.9%.
Based on the results, it was concluded 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: 0%: 1.0 ± 0.3 10%: 2.1 ± 0.7 25%: 6.4 ± 1.3 50%: 13.7 ± 3.4
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 0% (vehicle): 372 ± 71 10%: 772 ± 203 25%: 2392 ± 174 50%: 5085 ± 813

Any other information on results incl. tables

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

Auricular lymph nodes of control animals and animals treated at 10% were considered normal or small in size, whereas all lymph nodes of the animals treated at 25% and 50% were considered enlarged in size. The largest auricular lymph nodes were found in the highest dose group. No macroscopic abnormalities of the surrounding area were noted.

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.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
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 13.1% was calculated.
Based on these results:
- according to the recommendations made in the test guidelines, Terate® 091 Residue would be regarded as skin sensitizer.
- according to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, Terate® 091 Residue should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.
- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), Terate® 091 Residue should be classified as skin sensitizer (Category 1).
Executive summary:

Skin sensitisation of the test substance was determined in a guideline study (OECD TG 429) in mice (Local Lymph Node Assay). The test substance was found to be sensitising.