Tetrasodium [3-[(3-chloro-2-hydroxy-5-sulphophenyl)azo]-5-[[4-chloro-6-(3-sulphoanilino)-1,3,5-triazin-2-yl]amino]-4-hydroxynaphthalene-2,7-disulphonato(6-)]cuprate(4-)

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:

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: 18.83 – 20.99 g (at start of dosing), 19.57 – 19.89 g (in pilot experiment)- Housing: monitored conditions, microbiologically defined background, according to internal SOP No.40, animals in groups in macrolon cages with sterilized softwood shavings, cleaning and disinfection of animal room was regularly performed (according to internal SOP No.10)- 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: 21 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 3 °C (permanently monitored)- Humidity (%): 30 – 70 % (permanently monitored)- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

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 /mL)0.75 % w/v(7.5 mg /mL)

No. of animals per dose:

Pilot experiment: 3 femalesExposed groups: 15 females (5 animals in three groups)Positive control group: 5 femalesNegative control group: 5 femalesTotal: 28 animals

Details on study design:

PILOT TEST: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 (75, 7.5, 0.75 w/v). 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), MAIN STUDYDay 1 - 3: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.Days 4 - 5:No treatment.Day 6:The weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.76 x10+5 Bq of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed. TREATMENT PREPARATION AND ADMINISTRATION:The test substance was administered in the form of suspension in DAE 433 (mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol).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.

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 1

Key result

Parameter:

SI

Value:

4.27

Test group / Remarks:

75 %

Key result

Parameter:

SI

Value:

2.96

Test group / Remarks:

7.5 %

Key result

Parameter:

SI

Value:

2.95

Test group / Remarks:

0.75 %

Table 1: Individual Activities and Calculated Values

Group	Negative Control	Positive Control	75%	7.5%	0.75%
Activity (DPM)	395.79	3311.62	1201.84	836.06	922.63
	290.58	2222.54	1243.27	991.06	913.27
	254.27	3638.39	1391.05	1054.95	820.68
	283.01	2129.57	1371.30	941.54	1041.75
	321.30	4009.08	1385.73	755.17	851.74
mean	308.99	3062.24	1318.64	915.76	910.01
Standard Deviation	54.07	846.41	89.22	120.26	85.00
Stimulation Index	1.00	9.91	4.27	2.96	2.95

Table 2: Stimulation Index and ear weight

Group	Radioisotope incorporation		Ear weight
	Mean DPM	Stimulation Index	Mean (mg)
Negative Control	308.99	1.00	23.60
Positive Control	3062.24	9.91	40.14
75%	1318.64	4.27	25.16
7.5%	915.76	2.96	24.58
0.75%	910.01	2.95	24.88

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the given test conditions the animals exposed to the test substance, Reactive Violet 1, elicited sensitising response in LLNA assay. Positive results in cell proliferation revealed that the test substance, Reactive Violet 1, could be a contact allergen in mice. The test substance, Reactive Violet 1, provides positive 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 Reactive Violet 1 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 Reactive Violet 1 caused 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 highest treated group is > 3 (75 % - 4.27) and the value of disintegrations per minute (DPM) is significantly increased (at all dose levels) compared to negative control and overall a dose-effect relationship is observed.

The test substance did not cause marked increase of ear weight or other indication of irritation to skin at all dose levels nevertheless statistical significance was observed at the highest and the lowest dose levels. With regard to the Confidential test substance data sheet from Sponsor and no dose-response relationship 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.

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, Reactive Violet 1, provides positive sensitising response in LLNA assay. The SI at all dose levels was > 3. Dose-response relationship and statistical significance was observed simultaneously.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the LLNA test result, the test substance was classified as skin sensitising category 1B.