Registration Dossier

Administrative data

Description of key information

Skin sensitization (OECD 429), mouse: sensitising

Skin sensitiztion (OECD 406, Buehler), guinea pigs: sensitising

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:
08 - 21 Jun 2005
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)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in a closed container protected from heat, sparks and flame, protected from moisture or water, stored at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test material was soluble in the vehicle, N,N-Dimethylformamide (DMF).
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley Inc., MD, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 19.4 - 23.2 g
- Housing: animals were individually housed in suspended wire-mesh cages elevated over Bed-O´Cobs Combination bedding; animals were group housed for 5 h in a shoe-box tape cage (5 animals per cage) containing Bed-O´Cobs Combination bedding following tail vein injection of thymidine (3H-TdR); plastic tunnels were placed in each cage as an environmental enrichment device
- Diet: certified Rodent Diet #5002 (PMI International Inc.), ad libitum
- Water: municipal water, ad libitum
- Acclimation period: five days
- Indication of any skin lesions: baseline dermal score were recorded on study Day 1

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 - 22.5
- Humidity (%): 52 - 58
- Air changes (per hr): 10 - 12
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethylformamide
Concentration:
Range finding test:
- 10, 20, 40, 80 and 100% (v/v) in DMF
Main experiment:
- 20, 40 and 80% (v/v) in DMF
No. of animals per dose:
1 (range finding test)
5 (main test)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The test material was completely soluble in the vehicle (DMF). Test material concentrations of 10, 20, 40, 80 and 100% were used.
- Irritation: dermal scores were recorded on study Day 2 and 3.
- Ear thickness measurements: Ear thickness was measured on study Day 3.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: [3H]-methyl thymidine (3H-TdR) incorporation
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3H-TdR incorporation will be classified as a “non-sensitiser”.

TREATMENT PREPARATION AND ADMINISTRATION:
25 μl of the test item was applied to the dorsal surface of each ear of each mouse for three consecutive days; specifically, each day the dose was administered by applying a 12.5 µl aliquot to the left ear followed by an equal amount to the right ear. A further group of five mice received the vehicle or positive control alone in the same manner. Local irritation reactions were assessed. On day 6 an injection of 250 μl phosphate buffered saline (PBS) containing 20 μCi of [3H]-methyl
thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, the draining auricular lymph node of each ear was excised for each animal and pooled in a Stomacher sample bag containing 7 ml PBS. The lymph nodes were disaggregated by gentle mechanical disaggregation using a Stomacher 80 Lab System. The suspensions were centrifuged at 1000 rpm for 10 min at -4 °C. The pellets was re-suspended in 10 ml PBS and centrifuged again. Radioactive material was precipitated with 3 ml of 5% trichloroacetic acid at 4 °C for 18 hours. [3H]-TdR incorporation was measured by β-scintillation counting as disintegrations per minute (dpm).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data comparison was made using One Way Analysis of Variance (ANOVA) if the residues were normally distributed and the data had an equality of variance across the groups. Equality of variance was determined using Levene´s test. If the data did not satisfy the normality and equality of variance requirements, a Kruskal-Wallis test was performed. When an ANOVA or Kruskal-Wallis test was significant, pair-wise comparisons of the exposed groups or the positive control to the negative control were made using a Dunnett´s test or a Wilcoxon test, respectively. Using the disintegration per minute (dpm) data and the stimulation indexes, a quadratic regression on the dose levels was conducted using the data from the negative control and the 20, 40 or 80% test material groups. The resulting equation from the regression analysis for the stimulation index was used to determine the EC3 value.
Positive control results:
A group of five animals was treated with 25 μl of the positive control substance alpha-hexylcinnamaldehyde (HCA) as a 25% solution in DMF. A further control group of five animals was treated with DMF alone. The stimulation index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is 11.6. Therefore, alpha-hexylcinnamaldehyde (HCA) was considered to be a sensitiser under the conditions of the test.
Key result
Parameter:
SI
Value:
5.6
Test group / Remarks:
20% test material in DMF
Key result
Parameter:
SI
Value:
10.9
Test group / Remarks:
40% test material in DMF
Key result
Parameter:
SI
Value:
9.4
Test group / Remarks:
80% test material in DMF
Key result
Parameter:
EC3
Value:
7
Test group / Remarks:
test material
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
The stimulation index for each test group was calculated as followed: Stimulation index (SI) = mean dpm of test group / mean dpm of negative control group

CLINICAL OBSERVATIONS
Vehicle control animals as well as animals treated with 20 and 40% of the test material showed no notable observations. Animals treated with 80% test material showed no notable clinical observations on Day 1 and 2. Clinical observations were recorded on Days 3-6 and consisted of hair loss and flaking/scaling on the ears.
BODY WEIGHTS
The mean body weight for all groups indicated a mean body weight gain ranging from 1.62 to 4.83%.

DERMAL SCORE
Dermal scores on Day 6 were 0 for all groups except the group of mice treated with 80% test material where the dermal score was 2 (moderate irritation) for all animals in this group.

Table 1: Summary of LLNA result

Concentration (% in (v/v) DFM

mean dpm ± standard deviation

Stimulation index

Result

Vehicle

558.6 ± 308.7

1.0

Not applicable

20

3128.6 ± 2613.3

5.6

Positive

40

6086.8 ± 3463.8

10.9

Positive

80

5263.2 ± 1935.4

9.4

Positive

Positive control

6467.6 ± 2983.5

11.6

Positive

Interpretation of results:
other: CLP/EU GHS criteria are met, Category 1B classification is required according to Regulations (EC) No 1272/2008.
Conclusions:
The stimulation indices (SI) obtained in the LLNA conducted with the test material at concentrations of 20, 40 and 80% in DMF (v/v) were greater than 3 in each case (5.6, 10.9 and 9.4%, respectively) with statistical evidence of a dose-response and, hence the test material is considered positive for contact sensitisation. The EC3 value was calculated as 7%.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Two reliable in vivo skin sensitisation studies are available with the test substance (CAS 171869-89-9).

The key study is a LLNA test in mice according OECD 429 and in compliance with GLP (Dow Corning, 2005). The dose levels were selected based on an irritancy screen and were set to 20, 40 and 80%. The stimulation indices (SI) obtained with the test material at concentrations of 20, 40 and 80% in DMF (v/v) were greater than 3 in each case (5.6, 10.9 and 9.4%, respectively) with statistical evidence of a dose-response (Dow Corning, 2005). The stimulation indices were used to determine an EC3 value of 7%. Proper conduct of the study was verified by positive response (SI = 11.6) using α-hexylcinnamaldehyde as a positive control. Based on these results, the test substance is considered positive for contact sensitisation.

In a supporting study according to OECD 406 and in compliance with GLP the skin sensitising potential of the registration substance was assessed using guinea pig (Dow Corning, 2001). The method used was a modification of that described by Buehler, E.V. (1965), Delayed contact hypersensitivity in the guinea-pig. Based on the results of a preliminary study the test substance was applied as neat substance in the three induction applications as well as in the challenge application. In this study the test substance produced clear evidence of skin sensitisation in six animals of the twenty test animals (= 30%). Eight animals gave inconclusive responses and the remaining six of the twenty animals gave negative responses. As more than 15% of the animals gave positive responses, the test substance is considered to have the potential to cause skin sensitisation. All animals in the positive control group produced evidence of skin sensitisation following treatment with hexylcinnamaldehyde.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation meet the criteria for classification according to Regulation (EC) 1272/2008, and is therefore classified as Skin Sensitising Cat. 1B (H317).