Disodium 4-hydroxy-3-[[2-methoxy-5-methyl-4-[(4-sulphonatophenyl)azo]phenyl]azo]-7-(phenylamino)naphthalene-2-sulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

EC Number:

228-327-6

EC Name:

Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

Cas Number:

6227-20-9

Molecular formula:

C34H27N5O11S3.3Na

IUPAC Name:

trisodium;3-[[4-[(2E)-2-(6-anilino-1-oxo-3-sulfonatonaphthalen-2-ylidene)hydrazinyl]-5-methoxy-2-methylphenyl]diazenyl]naphthalene-1,5-disulfonate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Source: ENVIGO lab
Number of animals:
Pre experiment – 2 females
Tested group: 16 females (4 animals in four groups)
Negative control group – 1
Age: pre-experiment: 12 to 13 weeks, main test: 8-9 weeks
Body weight range: control group: 19 ± 0.8 g; tested groups: from 18.2 to 18.4 g
Health examination: All animals were examined during the acclimatisation period
Acclimatisation: 5 days

Animal rooms: Monitored conditions, microbiologically defined background, according to internal SOP No.40
Room temperature: 22 ± 2 °C
Relative humidity: 45-65 %,
Light: 12 hours light/dark cycle: 6am-6pm/6pm-6am
Animal caging: Animals in groups in Macrolon cages type II (pre test) and Type III (main test) with wire mesh top and granulated soft wood bedding.
Water: Drinking tap water ad libitum.
Diet: diet for experimental animals ad libitum (2018C Teklad Global 18% protein diet)

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0 % (v/v), only vehicle, negative control
5 % (v/v)
10 %(v/v)
25 % (v/v)

No. of animals per dose:

4 females per group

Details on study design:

Vehicle and Dose Selection: 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% suspension in PG.
Pre-test: To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days
Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ~3 was observed at any observation time and/or if an increase in ear thickness of ~25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of systemic toxicity. From day 1 to day 6 a possible redness of the ear skin could not or only with great difficulty (see day 3 animal 1) be determined due to the colour of the test item. On day 3, the animal treated with 10% test item concentration showed a very mild erythema of the ear skin (Score 1). Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

Positive control substance(s):

other: α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v)

Results and discussion

Positive control results:

5% dose: SI = 1.50
10% dose: SI = 3.84
25% dose: SI = 11.76
EC3: 8.2

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control group

Results 2

Parameter:

SI

Value:

2.99

Test group / Remarks:

5 %

Results 3

Parameter:

SI

Value:

3.29

Test group / Remarks:

10%

Results 4

Parameter:

SI

Value:

3.48

Test group / Remarks:

25%

Results 5

Parameter:

EC3

Value:

5.2

Test group / Remarks:

Test group at 5 and 10 % dose

Cellular proliferation data / Observations:

The EC3 value were 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.

Any other information on results incl. tables

In order to study a possible skin sensitising potential of the substance, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/w) in PG by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could technically be achieved. A control group of four mice was treated with the vehicle (PG) 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 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 determined by the incorporation of 3H - methyl thymidine measured in a â-scintillation counter.

The animals did not show any signs of systemic toxicity during the course of the study. From day 1 (1h after the first application) to day 6 a possible redness of the ear skin could not be examined due to the colour of the test item. On day 3 (1h before the third application), one animal (no. 14) belonging to the group treated with a test item concentration of 25% was unexpectedly found dead.

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 2.99, 3.29, and 3.48 (w/w) were determined with the test item at concentrations of 5, 10, and 25% in PG. A dose response was observed. The EC3 value calculated was 5.2% (w/w).

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Sensitizer for skin

Executive summary:

Under the test conditions, the animals exposed to the test substance showed some effect on skin.

The tested substance needs to be classified as sensitiser.