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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:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
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): Benzene, ethylenated, by-products from
- Physical state: Clear yellow liquid
- Analytical purity: Treated as 100% pure
- Lot/batch No.: AJ013 (50%), T3062-091609 (50%)
- Expiration date of the lot/batch: 30 October 2010 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: At room temperature in the dark; Stable under storage conditions

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J, inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: charles River France, L'Arbresle Cedex, France
- Age at study initiation:
- Weight at study initiation: 20-24 gms
- Housing: Individual housing in lable Macrolon cages (MI type; height 12.5 cm) containing sterlized sawdust as bedding material
- Diet: ad libitum, pelleted rodent diet (SM R.M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: ad libitum
- Acclimation period: At least 5 days before study start. Animals were group housed during the acclimation period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 º (actual range: 17.1 - 23.5 º)
- Humidity (%): 40-70 (actual range:21-67)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 21 December 2009 To: 11 January 2010

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary irritation study doses: 50% and 100%

Main study: 25, 50, or 100% (w/w); 0 (Acetone/Olive oil)
No. of animals per dose:
Preliminary irritation study: two young adult animals

Main study: Three groups of five animals
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: not applicable
- Irritation: 50%: left and right ear - erythema: 1, oedema 0; 50% right ear - erythema 1, oedema 0; 100% right ear - erythema 2, oedema 0
Based on the results, the highest test substance selected for the main study was a 1005 concentration.



MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: If the results indicated a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.


TREATMENT PREPARATION AND ADMINISTRATION:
The test subtance fromulations (w/w) were prepared within 4 hours prior to eaach treatment. No adjustment was made for specific gravity/density fo the test substance and the vehicle. Homogeneity was obtained to visually acceptable levels.

Induction - Days 1,2 and 3
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 test substance was spread over the entire dorsal surface ( Ø ~ 8 mm) of ear ear lobe. 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 was administered.

Observations
Mortality/Viability: Twice Daily
Toxicity: At least once daily
Body weights: On Days 1 (pre-treatment) and 6
Irriation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not applicable

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The SI values calculated for the substance concentrations 25, 50 and 100% were 2.6, 4.5 and 8.5 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 30.3% was calculated.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Due to possible outlier responses for individual animals within groups, median values were used to interpret the data. Median DPM/animal values for the esperimental groups treated with test substance concentration 25, 50 and 100% were 1074, 1851 and 3506 respectively. The median DPM/animal value for the vehicle control group was 412.

Any other information on results incl. tables

Based on these results:

- according to the recommendations made in the test guidelines, Benzene, ethylenated, by-products from would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007), Benzene, ethylenated, by-products from should be classified as skin sensitizer (Category 1).

- according to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, Benzene, ethylenated, by-products from should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The SI values calculated for the substance concentrations 25, 50 and 100% were 2.6, 4.5 and 8.5 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 subtance concentration that will give a SI = 3) of 30.3% was calculated.
Executive summary:

Test substance concentrations selected for the main study were based on the results of a preliminary study.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concnetrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 (v/v)).

Three days after the last exposure, all animals were injuected with 3H-methyl thymidine and after five hours, ear punches were weighed and the draining (auricular) lymph nodes were excised. After precipitating the DNA lymph nodes cells, radioactivity measurements were performed. The activity was expressed as the number of Distegrations per Minute (DPM) and a simulation index (SI) was subsequently calculated for each group.

Slight erythema was obsered for all animals at 25 and 50%. All animals at 100% showed slight to well-defined erythema combined with scabs behind the ears. No oedema was boserved in any of the animals examined. Mean ear weight/animal for the experimental groups treated with test substance concentrations 25, 50 and 100% were 32.10, 35.99 and 46.36 mg respectively. The mean ear weight/animal for the vehicle control group was 30.25 mg.

The majority of auricular lymph nodes were considered normal in size, except for the nodes in one animals at 25%, one animal at 50% and all animals at 100% which were considered enlarged. No macroscopic abnormalitites of the surrounding area were noted.

Due to possible outlier responses for individual animals within groups, median values were used to interpret the data. Median DPM/animal values for the experimantal groups treated with test substance concentrations 25, 50 and 1005 were 1074, 1851 and 3506 respectively. The median DPM/animal value for the vehicle control group was 412.

The SI values calculated for the substance concentrations 25, 50 and 100 were 2.6, 4.5 and 8.5, 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 30.3% was calculated.

The six monthly reliability check with Hexycinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriaate model for testing for contact hypersensitivity.