Registration Dossier

Administrative data

Description of key information

The skin sensitization potential of the test item was evaluated in a guinea pig maximization assay and in two human patch tests. All of them revealed negative results. In addition, a local lymph node assay in mice (OECD guideline 429, under GLP) on a structural analogue was performed. None of the tested concentrations induced a S.I. greater than the threshold value of 3. The test item (analogue) is not considered to be a skin sensitiser.

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:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 429 (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:
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 21-22g
- Housing: groups of five
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5d
- Fasting: 16h before administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethyl sulphoxide
Concentration:
2.5, 5 and 10%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25 % solution in dimethylsulfoxide. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
- Irritation: Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% (w/w) once daily each on three consecutive days. At the tested concentrations the animals did not show any signs of systemic toxicity. On day 6, the animal treated with 25% test item concentration showed an increase in ear thickness of 30% compared to pre-treatment value.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node assay
- Criteria used to consider a positive response:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the main study was assayed at 2.5, 5, and 10% (w/w) in DMSO. The different test item concentrations were prepared serially. The preparations were made freshly before each dosing occasion.

Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with different test item concentrations of 2.5, 5, and 10% (w/w) in dimethylsulfoxide. The application volume, 25 ul/ear/day, was spread over the entire dorsal surface ( ~8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine
3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytic, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application (day 6) 250 WL of phosphate-buffered saline (PBS) containing 19.5 WCi of 3HTdR (equivalent to 3HTdR 78 WCi/mL) were injected into each test and control mouse via the tail vein.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation). The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.
Positive control results:
substance: \-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1 v/v)
concentration: 0, 5, 10, 25%
DPM/lymph node: 382.2; 224.6; 577.4; 1424.4
S.I.: 1; 0.88; 1.51; 3.73
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle
Parameter:
SI
Value:
1.67
Test group / Remarks:
2.5 % group
Parameter:
SI
Value:
1.16
Test group / Remarks:
5 % group
Parameter:
SI
Value:
1.95
Test group / Remarks:
10 % group
Parameter:
other: disintegrations per minute (DPM)
Value:
826.1
Test group / Remarks:
vehicle group
Parameter:
other: disintegrations per minute (DPM)
Value:
1 379.3
Test group / Remarks:
2.5 % group
Parameter:
other: disintegrations per minute (DPM)
Value:
959.9
Test group / Remarks:
5 % group
Parameter:
other: disintegrations per minute (DPM)
Value:
1 607.1
Test group / Remarks:
10 % group

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Eventual erythema of the ear skin could not be evaluated due to the colour of the test item.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Procedure and results

The allergic potential of the substance was the topic of three publications. Naganuma et al (1983) described a guinea pig maximization assay where the test material was applied epicutaneously under occlusive condictions for induction and challenge exposure. The test material was considered to be a non-sensitizer. Concentration and puritiy of the test item was not mentioned. Moreover, two human patch tests were conducted. In the course of the first test (Sugai et al 1977), humans with cosmetic contact dermatitis who had already used product which contain the test material were exposed to the substance for 24h. 5 of 28 patients showed slight to moderate reactions. In the second test (Mitchell et al 1982) male and female humans were exposed for 48h to a 30% solution. None of the 50 people showed any skin reaction.

read across to CAS 1103 -39 -5

The test item as well as the structural analogue belong to the same category of metal laked pigments. All of the substances are of low solubility in water and octanol. The analogue substance is chosen because it is expected to be better soluble than the Barium and Strontium salts of this group. A detailed read cross justification is given in Annex I of the CSR.

In order to study a possible skin sensitising potential of the test item (analogue), three groups each of five female mice were treated once daily with the test item at concentrations of 2.5, 5, and 10% (w/w) in dimethylsulfoxide by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of five mice was treated with the vehicle (dimethylsulfoxide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a -scintillation counter. All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. Eventual erythema of the ear skin could not be evaluated due to the colour of the test item. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 1.67, 1.16 and 1.95 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in dimethylsulfoxide. A clear dose response was not observed. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. Thus, the test item is not considered to be a skin sensitiser under the test conditions of this study.

Discussion

A local lymph node assay on an analogue substance gives no indication for a skin senitizing potential. The human patch test performed by Mitchell et al was also negative for skin sensitization. The results of the patch test by Sugai et al are not assignable due to the pre-existing condition of the people, the very briefly decribed method, unknown dose level and unknown substance purity. In conclusion, the test substance is not considered to be sensitizing.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.