A mixture of: N-(4-chlorophenyl)-4-(2,5-dichloro-4-(dimethylsulfamoyl)phenylazo)-3-hydroxy-2-naphthalenecarboxamide; N-(4-chlorophenyl)-4-(2,5-dichloro-4-(methylsulfamoyl)phenylazo)-3-hydroxy-2-naphthalenecarboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP and OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA/CaOlaHsd
- Source: Harlan Netherlands
- Age at study initiation: Pre-Test 10-11 weeks (beginning of treatment); Main-Test 9-12 weeks (beginning of treatment)
- Weight at study initiation: Pre-Test: 22.3-24.2 g; Main-Test: 18.8-23.8 g
- Housing: single caging, Makrolon Type II (Pre-Test), Type III (Main-Test) with wire mesh top, granulated soft wood bedding
- Diet (e.g. ad libitum): 2018C Teklad global 18% protein rodent diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes (5 days prior to dosing)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 35 - 65 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 2.5, 5.0, 10.0% (w/w)
The highest concentration tested was the highest concentration that could be technically achieved.

No. of animals per dose:

4 animals per treatment group, 4 animals in the control group

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated for the body weight.

Where appropriate, the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

The ANOVA (Dunnett-test) was conducted on the ear weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group.

Results and discussion

Positive control results:

EC3: 9.3% (w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The S.I. values were 2.07, 2.75 and 2.62 for concentrations of 2.5, 5 and 10% in acetone/olive oil 4:1 (w/v) respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Results expressed as mean per group Group / DPM per lymph node / SI Neg. Control / 537.5 / 1.00 2.5% / 1113.5 / 2.07 5.0% / 1478.8 / 2.75 10.0% / 1408.6 / 2.62

Any other information on results incl. tables

No deaths, clinical signs, abnormal body weight development or relevant increase in ear weights were observed.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was found not to be a skin sensitiser in the LLNA when tested at 2.5, 5 and 10% in acetone/olive oil 4:1 (w/v).
The test item is not subject to classification and labelling.

Executive summary:

In order to study a possible skin sensitizing potential of the test item three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine; 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. 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 then determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. Due to the colour of the test item, redness of the ear skin could not be examined. A relevant increase in ear weights was not observed.

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 2.07, 2.75 and 2.62 were determined with the test item at concentrations of 2.5, 5 and 10% in acetone/olive oil (4+1, v/v). A 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.