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Administrative data

Description of key information

In a study to assess the skin sensitization potential using the local lymph node assay (LLNA), mice were exposed to TCD at concentrations of 10, 25 or 50 % v/v. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application.The SI obtained for 10, 25 and 50 % v/v were 1.2, 6.6 and 8.0 respectively which indicates that TCD showed the potential to induce skin sensitization.The EC3 value was determined to be 15.0 % v/v. The SI for the positive control substance hexyl cinnamic aldehyde was 7.3, which demonstrates the validity of this study.

TCD is regarded as a potential skin sensitizer.

Justification for selection of skin sensitisation endpoint:

One study available, considered to be valid and conducted in accordance with an accepted  test method under GLP.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April 2013 to 21 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: UK
- Age at study initiation: 80 - 012 weeks
- Weight at study initiation: 18.0 - 21.5 g
- Housing: 2/cage; solid-bottomed polycarbonate with stainless steel mesh lid
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 °C
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

IN-LIFE DATES: From: 11 April 2013 To: 20 May 2013
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 and 50% v/v
No. of animals per dose:
2 per group in preliminary phase
4 per group in main phase of study
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: a vehicle trial performed with TCD showed that it was miscible with 4:1 v/v acetone:olive oil (AOO) forming a clear colourless liquid which was satisfactory for dose administration.

- Irritation: preliminary investigations were performed to ensure the highest concentration to be used on the main study did not result in systemic toxicity or excessive local irritation. An assessment of local irritation was performed and an erythema score greater or equal to 3 and/or an increase in ear thickness of greater than 25% at Day 3 or 6 compared with predose would be regarded as irritation which precludes the use of that concentration on the main study.

- Lymph node proliferation response: not measured

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Method: animals were assigned without conscious bias
- Criteria used to consider a positive response: results for each treatment group were expressed as the Stimulation Index (SI). This was derived by dividing the mean dpm/mouse for each treated group and the positive control group by the mean dpm/mouse in the vehicle control group.
The positive control group is expected to give an SI of 3 or more to demonstrate the validity of the study.


TREATMENT PREPARATION AND ADMINISTRATION: the results of the preliminary investigations indicated that 50% v/v was a suitable high concentration for administration in the main phase of the study. The low and intermediate concentrations were selected from the concentration series given in regulatory guidelines and the concentrations administered on the main study were 10, 25 and 50% v/v in AOO.

Two female mice (per concentration) received a daily application of 25µL of appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The SI for the positive control substance hexyl cinnamic aldehyde was 7.3, which demonstrates the validity of the study.
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
6.6
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
8
Test group / Remarks:
50%

Preliminary test - mortality and clinical signs:

1) 10, 25 and 50 % v/v in AOO:

There were no deaths and no signs of ill health or toxicity were observed. Greasy fur on the head was noted following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

2) As supplied: One animal died approximately 90 minutes after dosing, signs of toxicity seen in both mice included, under activity and hunched posture. Greasy fur on the head was noted following dosing, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance No erythema was observed on the ears at all levels on Days 1 to 6. There was no evidence of an effect of treatment on ear thickness at all levels. There was no indication of an overt effect of treatment on bodyweight gain. A loss in bodyweight was noted over the study period for one mouse dosed but is not considered to be an effect of treatment. Both mice dosed with the test material as supplied lost weight between the start of treatment and death. On the basis of the results from the preliminary investigation, 50 % v/v was considered a suitable high concentration for administration in the main phase of the study.

Main study

Mortality and clinical signs:

There were no deaths and no signs of ill health or toxicity were observed during this study. Partially closed eyelids were noted for one mouse in the vehicle control group, three mice dosed at 50 % v/v and one mouse in the positive control group. This sign was noted shortly following dosing on Day 2 only for each of these animals. As the observation was evident in the vehicle and positive control animals it was not test substance related. Greasy fur on the head was noted for all animals following each dosing occasion, this was related to unoccluded dermal administration of an oily vehicle. No signs of dermal irritation were seen on the ear during the study. There was no indication of an effect of treatment on bodyweight gain. A loss in bodyweight was noted for three mice in the positive control group over the study period. A small loss in bodyweight is not uncommon in young laboratory mice and as the positive control group responded in the expected manner this bodyweight loss was not considered to be toxicologically significant.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
TCD is regarded as a potential skin sensitizer. The EC3 value was calculated to be 15.0 % v/v.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The EC3 value for TCD was determined to be 15.0. According to the CLP Regulation 3.4.2.2.3.1 a stimulation index (SI) of three or more is considered a positive response in the local lymph node assay and Category 1 classification applies.

In accordance with Table 3.4.3 of the CLP Regulation, TCD can be classified as sub-category 1B on the basss that the EC3 value is greater than 2 %.