Calcium bis[2-[(2-hydroxynaphthyl)azo]naphthalenesulphonate]

Endpoint summary

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well described, GLP and OECD guideline conform

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Remarks on result:

other: vehicle group: 1.00 2.5% group: 1.67 5% group: 1.16 10% group: 1.95

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: vehicle group: 826.1 2.5% group: 1379.3 5% group: 959.9 10% group: 1607.1

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:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In order to study a possible skin sensitising potential of the test item, 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.

Migrated from Short description of key information:
In order to study a possible skin sensitising potential of the test item, 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 (according to OECD guideline 429, under GLP conditions). 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.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin sensitization under Directive 67/548/EEC.

                               

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.