Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 17 th to 30 th, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with no significant deviations or restrictions.

Data source

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

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): α, α, α, trichlorotoluene
- Physical state: clear colourless liquid
- Analytical purity: 99.21 %
- Lot/batch No.: S100405003
- Expiration date of the lot/batch: 12 April 2011 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under test conditions:
Stability in vehicle:
• Acetone: Not indicated
• Olive oil: Not indicated
Solubility in vehicle:
• Acetone: Not indicated
• Olive oil: Not indicated
- Storage condition of test material: At room temperature in the dark

No further infiormation

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Horst, The Netherlands.
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Housing: Animals were housed in a controlled environment individually in Macrolon cages
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): a temperature of 21.0 ± 3.0ºC (actual range: 19.7 - 22.9ºC)
- Humidity (%): a relative humidity of 40-70% (actual range: 43 - 62%)
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

No further information

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Group animal numbers induction (test substance; % w/w)

1 01 - 05 0 (Acetone/Olive oil (4:1 v/v))
2 06 - 10 1
3 11 - 15 5
4 16 - 20 10

No. of animals per dose:
Five animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Containers with pre-weighed test substance or formulations were wrapped with thin foil. No adjustment was made for specific gravity or density of the vehicle or test substance. Homogeneity was obtained to visually acceptable levels.
- Irritation: A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2at the highest concentration.

Initially, two test substance concentrations were tested; a 100% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

Based on the results of the initially treated animals, three additional animals were treated in a similar manner with three lower concentrations (5%, 10% and 25%) at a later stage

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- 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

No further information
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No thorough statistics needed

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 1, 5 and 10% were 7.2, 15.9 and 19.5 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 1, 5 and 10% were 8538, 18776 and 23082 DPM respectively. The mean DPM/animal value for the vehicle control group was 1182 DPM

Any other information on results incl. tables

The slight irritation of the ears as shown by some animals treated at 1 and 5% and the slight to well-defined irritation in all animals treated at 10% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

All nodes of the test substance treated animals were increased in size. 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.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 CLP and DSD criteria
Conclusions:
According to the authors, these results show that the test substance elicits a stimulation index (SI) higher than 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was not determined. Thus, based on these results, according to the recommendations made in the test guidelines and according to the criteria laid down in Regulation (EC) No 1272/2008 and in the DSD regulation (EEC) No 67/548, α, α, α, 4-tetrachlorotoluene would be regarded as skin sensitizer.
Executive summary:

The authors of this study report conducted the assessment of Contact Hypersensitivity to α, α, α, 4 -tetrachlorotoluene in the Mouse (Local Lymph Node Assay). The study was carried out according to the OECD guideline n° 429 with no observed deviations or restrictions.

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

In the main study, three experimental groups of five female/J mice were treated with test substance concentrations of 1, 5 or 10% 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 injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating theof 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.

 

No oedema was observed in any of the animals examined.

 

All nodes of the test substance treated animals were increased in size. 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.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.

 

Mean DPM/animal values for the experimental groups treated with test substance concentrations 1, 5 and 10% were 8538, 18776 and 23082 DPM respectively. The mean DPM/animal value for the vehicle control group was 1182 DPM.

 

The SI values calculated for the substance concentrations 1, 5 and 10% were 7.2, 15.9 and 19.5 respectively. These results show that the test substance elicits an SI3. The EC3 value (the estimated test substance concentration that will give a SI =3) was not determined.

 

The six-month reliability check with α-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate and valid model for testing for contact hypersensitivity.

Based on these results, according to the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures and the DSD regulation (EEC) No 67/548, α, α, α, 4 -tetrachlorotoluene should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction or R 43: May cause sensitisation by skin contact according to the DSD directive.