Disodium; 4-Amino-3-{4-[4-(2,4-diamino-phenylazo)-benzenesulfonylamino]-phenylazo}-5-hydroxy-6-phenylazo-naphthalene-2,7-disulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

disodium 4-amino-3-[2-(4-{4-[2-(2,4-diaminophenyl)diazen-1-yl]benzenesulfonamido}phenyl)diazen-1-yl]-5-hydroxy-6-(2-phenyldiazen-1-yl)naphthalene-2,7-disulfonate

EC Number:

605-104-5

Cas Number:

157577-99-6

Molecular formula:

C34H26N10Na2O9S3

IUPAC Name:

disodium 4-amino-3-[2-(4-{4-[2-(2,4-diaminophenyl)diazen-1-yl]benzenesulfonamido}phenyl)diazen-1-yl]-5-hydroxy-6-(2-phenyldiazen-1-yl)naphthalene-2,7-disulfonate

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

Experimental Animals
Animals species and strain: Young adult female mice (nulliparous and non-pregnant), strain BALB/CBYJICO (referred to as BALB/c)
Source: Breeding farm VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
Number of animals per Group:
Pilot experiment - 3 females
Exposed groups – 15 females (5 animals in three groups)
Positive control group – 5 females
Negative control group – 5 females
Total: 28 animals
Body weight range:
In pilot experiment: 17.14-18.27 g
In main test: 16.01 - 19.56 g (at start of dosing)
Health examination: All animals were examined during the acclimatisation period
Acclimatisation:
pilot experiment: 6 days
main experiment: 13 days

Animal quarters/husbandry
Animal rooms: Monitored conditions, microbiologically defined background, according to internal SOP No.40
Microclimatic conditions: Room temperature: 22 ± 3 °C, permanently monitored
Relative humidity: 30 – 70 %, permanently monitored
Light: 12 hours light/dark cycle: 6am-6pm/6pm-6am
Animal caging: Animals in groups in macrolon cages with sterilized softwood shavings
Water: Drinking tap water ad libitum. Water quality corresponded to Regulation No. 252/2004 Czech Coll. Of Law, Health Ministry
Diet:Pelleted standard diet for experimental animals ad libitum (Altromin, manufacturer: Altromin Spezialfutter GmbH & Co KG, Germany). Microbiological control and content of nutrients is performed according internal SOP No. 72
Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed, as it is described in internal SOP No.10

Animal identification
Cage identification: By cage number, study number and group specific colour.
Animal identification: By felt tip marking (from 1 to 5 per each group and group specific colour)
Random selection: According to internal SOP No.42

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 – mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol

Concentration:

30%, 3%, 0.3% test item concentrations in the form of suspension in DAE 433

No. of animals per dose:

5 animals per dose

Details on study design:

Pilot experiment
The test item was administered to three animals (one animal per group) to assess a possible systemic toxicity or high irritation of the ears skin. The appropriate suspensions (25 uL) of the test item (30%, 3.0%, 0.3% w/v) were applied to three animals (dorsum of each ear) every morning for 3 consecutive days. Both ears of each mouse were observed for erythema and scored and subsequently ear thickness was measured using digital thickness gauge.

According to the result of pilot experiment, the following test item concentrations doses were chosen for main study:
Concentrations of test item in application forms:
30% (w/v) 300 mg/mL
3% (w/v) 30 mg/mL
0.3% (w/v) 3 mg/mL

Main study
The test item was administered to the dorsum of each ear of mice once a day for 3 consecutive days.
Three concentrations were used in pilot experiment: 30%, 3%, 0.3% concentrations in the form of suspension in DAE 433.
In the main study, three treated groups were included: 30%, 3%, 0.3% test item concentrations in the form of suspension in DAE 433.

At the end of the experiment, the incorporation of 3H-methyl thymidine to tissue of auricular lymph nodes was measured by scintillation counting.
The Stimulation Index (for incorporation of 3H-methyl thymidine) was calculated from values of exposed and control groups.

Demonstration of test efficiency
Experiment for the demonstration of test efficiency was included in the study:
A group of animals treated with a substance with proven positive effect was included in the test (positive effect = Stimulation Index SI ≥ 3). The substance DNCB (dinitrochlorobenzene, 0.5% (w/v) solution) was used as the positive control. A dose level (25μL) for DNCB was determined during the verification of the method

Positive control substance(s):

other: dinitrochlorobenzene, 0.5% (w/v) solution)

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XVII, USA) was used. Statistical evaluation of measured parameters was performed by applying the parametric test for testing whether all group samples originate from the same distribution and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons

Results and discussion

Positive control results:

The positive control substance, DNCB, produced a positive LLNA response at the exposure level expected to give an increase in the Stimulation Index SI ≥ 3 over the negative control group, which was in agreement with the expected mode of action of a contact allergen (SI=10.17). The positive control also elicited a reaction pattern with significant increase in ear weight. The negative control did not show any changes. These results demonstrate that the method performed in the conditions of the laboratory has sufficient reliability

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.05

Test group / Remarks:

30 %

Results 2

Parameter:

SI

Value:

1.04

Test group / Remarks:

3 %

Results 3

Parameter:

SI

Value:

1.17

Test group / Remarks:

0.3 %

Cellular proliferation data / Observations:

The SI for the 30% (1.05), 3% (1.04) and 0.3% (1.17) test item groups was below the threshold, and the stimulation index (SI) is < 3.

Any other information on results incl. tables

Body Weight and Body Weight Increments

The statistical analysis was performed for body weight at the 1st and 6th day of study. Statistically significant differences were not found.  The body weights of treated groups and the positive control group were similar compared to the negative control animals during the whole study.

Body weight increment was calculated from values of day 6 just before necropsy and day 1 before first application. Body weight increments were decreased at the dose levels 30% and 0.3%.

Mortality, Clinical Observations

No animals died during the main study. No symptoms of toxicity and no erythema on the application site were observed in all animals from the 30%, 3% and 0.3% test item groups.

All animals in the positive control group showed symptoms caused by the application of DNCB: hyperemia of auricle, clonic spasm and increased response to stimuli.

Irritating Effect of the Test Item

In the positive control group, the ear weight was statistically significantly increased compared to the negative control group.

No statistically significant changes of ear weight were recorded in the 30%, 3% and 0.3% test item groups.

Evaluating the Redness of the Ear Skin

Because ears of the animals were slightly colored with the test item, the evaluation of redness was performed up to four hours after application.

No erythema of the skin was observed during the clinical observation at 30%, 3% and 0.3% dose levels.

Very slight erythema and well-defined erythema in animals of the positive control group were recorded during the study

Applicant's summary and conclusion

Interpretation of results:

other: not skin sensitizing

Conclusions:

Under the given test conditions, the test item, Acid Black 234, does not elicit sensitising response in LLNA.

Executive summary:

The test item, Acid Black 234, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay.

The Local Lymph Node Assay (LLNA) with the incorporation of 3H-methyl thymidine radionuclide was used. The testing was conducted according to the Method B.42 – Skin Sensitisation: Local Lymph Node Assay, Commission Regulation (EU) No.640/2012, published in O.J. L 193, 2012 and OECD Test Guideline No. 429, Skin sensitisation: Local Lymph Node Assay, Adopted 22th July 2010.

In the pilot experiment, the following concentrations of the test item in the vehicle DAE 433 were used: 30%, 3%, and 0.3%. Based on the results, the following dose levels were selected for main study: 30%, 3%, and 0.3%.

In this study the contact allergenic potential of Acid Black 234 was evaluated after topical application to female BALB/c mice. Mice were exposed to three concentrations of the test item in vehicle DAE 433 (30%, 3% and 0.3%) for 3 consecutive days. The positive control item was Dinitrochlorobenzene (DNCB).

Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index (SI), was determined. The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

There were no mortalities. No statistically significantly changes in ear weight were recorded at the 30% 3% and 0.3% dose levels, compared to the negative control. The animals exposed to 30%, 3% and 0.3% Acid Black 234 showed no skin reactions and no other negative clinical symptoms of intoxication throughout the experiment.

The positive control item, Dinitrochlorobenzene (DNCB), as a known contact allergen (0.5% (w/v)) elicited the expected reaction pattern with a significant increase in the Stimulation Index (10.17) and ear weight. Appropriate performance of the assay in the test laboratory was therefore demonstrated.

The DPM values were no statistically significantly changed in animals dosed at 30%, 3% and 0.3% compared to the negative control. The values of the SI for groups treated at the 30% (1.05) 3% (1.04) and 0.3% (1.17) dose levels were below the threshold.

The test item, Acid Black 234, no caused significant change in radioisotope incorporation into the DNA of dividing lymphocytes, so the result of LLNA assay is negative.

Under the given test conditions, the test item, Acid Black 234, demonstrated a negative sensitising response in the LLNA assay. The SI at all dose levels (30%, 3% and 0.3%) was ≤3. Negative results in cell proliferation revealed that the test item Acid Black 234 is not a contact allergen in mice.