Disodium 2,2'-(azodi-p-phenylene)bis[6-methylbenzothiazole-7-sulphonate]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Council Regulation (EC) No.640/2012, published in O.J. L 193 (2012)

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:

Disodium 2,2'-(azodi-p-phenylene)bis[6-methylbenzothiazole-7-sulphonate]

EC Number:

233-311-7

EC Name:

Disodium 2,2'-(azodi-p-phenylene)bis[6-methylbenzothiazole-7-sulphonate]

Cas Number:

10114-47-3

Molecular formula:

C28H20N4O6S4.2Na

IUPAC Name:

disodium 2,2'-(diazene-1,2-diyldi-4,1-phenylene)bis(6-methyl-1,3-benzothiazole-7-sulfonate)

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

Trade name: Saturn Yellow LFF 200Batch No.: 4003/S

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Breeding farm VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic- Females (if applicable) nulliparous and non-pregnant: yes- Microbiological status of animals, when known: All animals were examined during the acclimatisation period- Age at study initiation: 8 to 10 weeks (at start of dosing)- Weight at study initiation: 15.70 - 17.98 g (at start of dosing), in pilot experiment 15.69 - 16.39 g- Housing: Animals in groups in macrolon cages with sterilized softwood shavings, monitored conditions, microbiologically defined background- Diet (e.g. ad libitum): Pelleted standard diet for experimental animals ad libitum (microbiological control and content of nutrients)- Water (e.g. ad libitum): drinking tap water ad libitum- Acclimation period: 7 days- Indication of any skin lesions: yesENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 3 °C, permanently monitored- Humidity (%):30 – 70 %, permanently monitored- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 (dimethylacetamide - acetone - ethanol 4 : 3 : 3)

Concentration:

0.5% w/v (5 mg /mL) 5% w/v (50 mg /mL)50% w/v (500 mg/mL)

No. of animals per dose:

5 animals per dose

Details on study design:

PRE-SCREEN TESTS:The test substance was administered to three animals to assess a possible systemic toxicity or high irritation of skin. One animal per dose group was used in pilot experiment. The test substance was administered in the form of suspension in DAE 433. The pilot experiment was conducted under the conditions identical to the main study, except the assessment of lymph node proliferation. The appropriate suspensions of the test substance (50%, 5%, 0.5% w/v) was applied to three animals in volume 25 µl to the dorsum of each ear once a day morning for 3 consecutive days. The suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application. The application was performed very slowly by micropipette. The route of administration was the same as in the main study. Body weight was recorded before application and prior to termination (Day 6).Both ears of each mouse were observed for erythema and scored and subsequently ear thickness was measured using digital thickness gauge. Immediately after scheduled humane kill of animals, the both ears were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm (=0.5 cm2) were excised using a disposable punch and weighed together on analytical scales.MAIN STUDY Animal Check-in and Allocation to GroupsAnimals were subjected to a clinical examination (health check) shortly after arrival. No clinical changes were recorded. After acclimatization the animals have been randomly allocated to the dose groups and assigned animal numbers. Application The test substance was administered in the form of suspension in DAE 433. The volume of the application form was constant at all groups of animals - 25 µl of the appropriate suspension to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette. Losses caused by draining from the ear must be minimized. The application forms of test substance (suspensions) were prepared immediately before administration. Experimental Schedule Day 1:Open application of 25 μL (in the morning, by pipette) of appropriate suspension of the test substance, the vehicle alone or the positive control to the dorsum of each ear.Days 2 and 3:The application procedure repeated as carried out on day 1.Days 4 and 5:No treatment.Day 6:The weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.881×10^5 Bq of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed. In Vivo Examination Mortality During working hours, the animals were checked for general health whenever other activities were performed – twice daily during the dosing period. Clinical ObservationsClinical observation was performed twice daily during the dosing period. All changes in behaviour and health condition of animals were recorded. E.g.: piloerection, lacrimation, appearance of skin, fur and mucous membrane, ataxia, tremors, and convulsions, aggressiveness, change in grooming activity, marked change in activity level changes in frequency and intensity of breathing such as dyspnoea, gasping, and rales etc.Efforts were made to characterize onset and duration of signs observed. During clinical observations the examination of skin irritation at application site was carried out and irritation score was recorded. Body WeightIndividual body weights were measured using an electronic balance. Weighing was performed on the first day of treatment, before dosing, and on the day of necropsy before application of the radionuclide. Necropsy The third day after last administration (five hours after application of radionuclide), all test animals were sacrificed by prolonged ether narcosis. Post Mortem Investigations Ear WeightsImmediately after scheduled humane kill of animals, the both ears were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm (=0.5 cm2) were excised using a disposable punch and weighed together on analytical scales. Incorporation of 3H-methyl ThymidineThe tissue of both of the lymph nodes was prepared by gentle mechanical disaggregation through 100 m-mesh nylon gauze with concomitant pooling in 1 mL PSB (Phosphate Buffered Saline). The pairs of lymph nodes were analysed separately for each animal per group. Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4°C for 18 h. The pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine was measured by β-scintillation counting (Beckmann LS 6500) as disintegrations per minute (DPM).

Positive control substance(s):

other: 2,4-dinitrochlorobenzene (CAS: 97-00-7)

Statistics:

For statistical calculations, the software Statgraphic Centurion (version XV, 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.Statistical evaluation of the body weightAs the first step the test for normality (Shapiro-Wilk test) was used. Since the smallest P-value amongst the tests performed is lower than 0,05, we can reject the idea that data comes from a normal distribution with 95% confidence. The transformation of data was performed (Box-Cox transformation). Because the normal distributed distribution was achieved after transformation of data then the One-Way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means. Statistical evaluation of ears weightsAs the first step the test for normality (Shapiro-Wilk test) was used. Since the smallest P-value amongst the tests performed is lower than 0,05, we can reject the idea that data comes from a normal distribution with 95% confidence. The transformation of data was performed (Box-Cox transformation). Because the normal distributed distribution was not achieved after transformation of data then the non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used. Statistical evaluation of DPM (Desintegration Per Minute)Non-parametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons was used for statistical evaluation of the value of DPM.

Results and discussion

Positive control results:

All animals in the positive control group showed symptoms caused by the application of DNCB: hyperaemia of skin with well defined erythema on application site, clonospasm and increased response to stimuli.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.18

Test group / Remarks:

50 %

Results 2

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

5 %

Results 3

Key result

Parameter:

SI

Value:

0.91

Test group / Remarks:

0.5 %

Cellular proliferation data / Observations:

The value of DPM and SI for positive control group was increased. The SI was ≥ 3 (5.57) – the LLNA was efficient. The SI for the test groups treated by the test substance at the all dose levels was below the threshold, stimulation index (SI) is < 3. Statistically significant increase of DPM for highest dose level was observed but value of DPM and SI was almost identical with values of negative control.The EC3 value, calculated using the smallest squares method, is higher than 100 %.

Any other information on results incl. tables

Table 1: Individual Activities, Stimulation Index and Ear weights

Group	NC	PC	50%	5%	0.5%
Individual Activity (DPM)	493.21	3191.50	630.13	533.91	482.36
	521.53	2791.09	629.06	469.89	766.04
	535.33	2478.21	625.05	495.10	423.33
	613.31	3651.30	607.72	729.41	394.47
	502.65	2750.26	648.37	458.89	367.08
mean	533.21	2972.47	628.07	537.44	486.66
Stimulation Index	1.00	5.57	1.18	1.01	0.91
Ear weight (median) (mg)	25.60	43.30	27.00	26.00	25.40

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given test conditions, the animals exposed to the test substance, Direct Yellow 28, do not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Direct Yellow 28 could not be a contact allergen in mice.The test substance Direct Yellow 28, provides negative sensitising response in LLNA assay.

Executive summary:

The test substance, Direct Yellow 28, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test substance health hazard evaluation.

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

In this study the contact allergenic potential of Direct Yellow 28 was evaluated after topical application to female BALB/c mice. Mice were exposed to three concentrations of test substance suspended in vehicle DAE 433 (mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol) for 3 consecutive days.

In pilot experiment the following concentrations of test substance in application forms were used: 50 %, 5 %, 0.5 % (w/v). According to the results of pilot experiment the same doses were confirmed for main study.

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, was determined. The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

The animals exposed to the test substance at all doses showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment.

Residues of the test substance on the ears were visible during whole study so it could cause slight weight increase detected only at the highest dose level. So, no irritation effect was observed which could impair the result of LLNA assay.

Stimulation Index at the all dose levels was < 3 so the result of LLNA assay is negative (see criteria in chapter 3.8. of this report).

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

Under the given test conditions, the animals exposed to the test substance, Direct Yellow 28, do not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Direct Yellow 28 could not be a contact allergen in mice.

The test substance Direct Yellow 28, provides negative sensitising response in LLNA assay.