Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.42 (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:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Breeding farm VELAZ, Unětice 139, 252 62, Czech Republic (RČH CZ 21760118)- Age at study initiation: 8 - 10 weeks- Weight at study initiation: 18.14 – 21.66 g (in pilot experiment 18.27 – 19.16 g)- Housing: in groups in macrolon cages with sterilized softwood shavings at monitored conditions, microbiologically defined background (according to internal SOP No.40)- Diet (e.g. ad libitum): 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- Water (e.g. ad libitum): drinking tap water ad libitum (water quality corresponded to Regulation No. 252/2004 Czech Coll. Of Law)- Acclimation period: 28 daysENVIRONMENTAL CONDITIONS- Temperature: 22 ± 3 °C, permanently monitored- Humidity: 30 – 70 %, permanently monitored- Photoperiod: Light: 12 hours light/dark

Vehicle:

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

Concentration:

75% (w/v) - 750 mg/mL 7.5% (w/v) - 75 mg /mL0.75% (w/v) - 7.5 mg /mL

No. of animals per dose:

3 animals per dose

Details on study design:

RELIABILITY CHECK For the demonstration of test efficiency a group of animals treated with a substance with proved positive effect is included in each 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 for DNCB was determined during the verification of the method. The vehicle and dosage volume were the same as in treated groups. An application form was prepared on each day of administration by dissolving an appropriate amount of the positive control substance in the vehicle to obtain a concentration of 5 mg/mL. The solution was mixed for 5 minutes with a magnetic stirrer. PILOT EXPERIMENTThe 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 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 (75%, 7.5%, 0.75% 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. Both ears of each mouse was observed for erythema and scored and subsequently ear thickness was measured using digital thickness gauge. Body weight was recorded before application and prior to termination (Day 6).According to the results of pilot experiment, the doses used in pilot experiment were chosen also for main study.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 l 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 25μL (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.- Day 2 and 3: The application procedure repeated as carried out on day 1.- Day 4 and 5: No treatment.- Day 6: The weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.76 e5 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 Observations: Clinical observation was performed according to internal SOP M/1 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.- Body Weight: Individual 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.POST MORTEM INVESTIGATIONS- Ears Weights: 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. - Incorporation of 3H-methyl Thymidine: The tissue of both of the lymph nodes was prepared by gentle mechanical disaggregation through 100 µm-mesh nylon gauze with concomitant pooling in 1 mL PSB (Phosphate Buffered Saline). The pairs of lymph nodes was 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 18h. 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).DATA TREATMENT Mean values and standard deviations of ears weight and incorporation of 3H-methyl thymidine were computed for the test substance groups and for the positive as well as the vehicle control group. Stimulation index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from exposed groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control group was set at 1 by definition.

Positive control substance(s):

other: 1-chloro-2,4-dinitrobenzene (CAS 97-00-7)

Statistics:

For statistical calculations the software Statgraphic Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis 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.

Positive control results:

See Table No. 1

Key result

Parameter:

SI

Value:

1.24

Test group / Remarks:

0.75 %

Key result

Parameter:

SI

Value:

1.12

Test group / Remarks:

7.5 %

Key result

Parameter:

SI

Value:

1.29

Test group / Remarks:

75 %

Cellular proliferation data / Observations:

EC3 CALCULATIONCalculadted using least squares method; EC3 < 100 %.CLINICAL OBSERVATIONS:No animal died during the main study.Just reduction of body weight at the highest dose level but no other symptoms of toxicity were observed during the study. No erythema on application site was observed in all animals from the negative control group and all animals administered by the test substance.All animals in the positive control group showed symptoms caused by the application of positive control substance: hyperaemia of skin with well defined erythema on application site, clonospasm and increased response to stimuli. BODY WEIGHTSReduction of body weight was recorded in all animals at the highest dose level. One female has markedly reduced its weight (-2.11 g), in other females the reduction of body weight in tenths of grams was observed. Body weight increment was calculated from values of day 6 just before necropsy and day 1 before first application. Body weight increment was lower in all treated groups. The lowest increment was at the highest dose level.

Table No. 1: Results of Main Study

Group	negative control	positive control	75%	7.5%	0.75%
Activity (disintegrations per minute)	132.20	2171.00	183.50	156.02	138.91
	102.16	1687.32	132.56	160.75	143.53
	124.05	1196.97	154.70	161.18	185.24
	160.81	1868.14	131.65	127.82	120.22
	126.77	1544.89	229.71	120.80	213.10
mean	129.20	1693.66	166.42	145.31	160.20
standard deviation	21.05	363.03	41.20	19.44	37.93
stimulation index	1.00	13.11	1.29	1.12	1.24

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given test conditions the animals exposed to the test substance, Direct Black 51, does not elicit sensitising response in LLNA assay. Positive results in cell proliferation revealed that the test substance, Direct Black 51, could not be a contact allergen in mice. The test substance, Direct Black 51, provides negative sensitising response in LLNA assay.

Executive summary:

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

In this study the contact allergenic potential of Direct Black 51 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: 75 %, 7.5 %, 0.75 % (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.

Comparison of Stimulation Indexes between treated groups and control vehicle group revealed that the test substance Direct Black 51 did not cause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes of animals treated by the test substance at all concentrations. The Stimulation Index of the all treated groups is < 3 and the value of disintegrations per minute (DPM) is not significantly increased compared to negative control.

The test substance caused slight increase of ear weight but no other indication of irritation to skin at all dose levels nevertheless statistical significance was observed at the highest dose level. With regard to the negative results of the study No.: 270/15/4AI: Direct Black 51- In vitro Skin Irritation Test (EpiDermTM Model); VUOS-CETA Report No. 15-323, 2015 it was not possible that irritation of skin was caused. Residues of the test substance on the ears were visible during whole study so it could cause this weight increase.

The animals exposed to the test substance at all doses showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment. Only reduction of body weight was observed at the highest dose level.

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 test substance, Direct Black 51, provides negative sensitising response in LLNA assay. The SI at all dose levels was < 3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on available LLNA test result, the test substance does not cause skin sensitisation. No respiratory sensitisation data is available. Direct Black 51 does not meet the criteria for classification according to Regulation (EC) No. 1272/2008.