Trisodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)][2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzenesulphonato(3-)]chromate(3-)

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:

other: read across from similar substance

Adequacy of study:

key study

Study period:

March from 18th to 31st, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines and performed according to GLP. Justification for Read Across is detailed in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22nd July 2010

Deviations:

yes

Remarks:

not affecting the study results

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
- Justification for the strain tested: the BALB/c strain is selected because of the availability of historical control data for this strain at this facility and because it was regarded as a sensitive strain in the context of contact allergy (Woolhiser et al., 2000; Ehlinga et al., 2005).
- Source: breeding farm VELAZ s.r.o., Unětice 139, 252 62, Czech Republic, RČH CZ 21760118
- Age at study initiation: young adult mice (nulliparous and non-pregnant), 8 to 10 weeks old.
- Weight at study initiation: 17.96 – 19.42 g, in pilot experiment 18.41 – 18.61 g.
- Housing: maximum six in macrolon cages (35x20x15 cm) with sterilized softwood shavings. Cleaning and disinfection of animal room was regularly performed, as it is described in internal SOP No.10. It followed the two-stage process: mechanical cleaning and disinfection own. It is used prescribed concentration of disinfectants and the exposure time.
- Diet: pelleted standard diet for experimental animals ad libitum (ST - 1, VELAS a.s., Hrabanov 535, 289 22 Lysá nad Labem, CZ 801080-01).
- Composition of diet: Wheat, Oats, Fish meal powder, Dried snail-clover, Soya extracted Gross, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex.
- Nutrition complex: crude protein - min. 21 %, Drip - max. 14 %, Fat-min. 3 %, Fiber- max. 4.1 %, Ash-max. 7 %, Calcium- min. 1 %, Phosphorus-min 0.8 %, Magnesium -min. 0.2 %, Sodium-max. 0.25 %.
- Water: drinking tap water ad libitum.
- Acclimation period: 15 days; all animals were examined during the acclimatisation period.

ENVIRONMENTAL CONDITIONS
- Animal rooms: monitored conditions, microbiologically defined background, according to internal SOP No.40. Control of animal rooms is performed four times a year and animals twice a year in accredited testing laboratory according actual FELASA recommendation.
- Temperature: 22 ± 3 °C, permanently monitored.
- Humidity: 30 – 70 %, permanently monitored.
- Photoperiod: 12 hours light/dark cycle, i.e. 6am-6pm/6pm-6am.

Vehicle:

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

Concentration:

50 % (w/v) 500 mg/ml; 5 % (w/v) 50 mg/ml; 0.5 % (w/v) 5 mg/ml

No. of animals per dose:

Pilot experiment: 3 females
Main study: 5 animals x dose x group

Details on study design:

RANGE FINDING 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.
- Concentrations of test substance: the test substance was administered in the form of suspension in DAE 433. 50 % (w/v) 500 mg/ml; 5 % (w/v) 50 mg/ml; 0.5 % (w/v) 5 mg/ml.
- Suspension preparation: the suspensions were prepared before the start of application by mixing on magnetic stirrer and then were still mixed during application.
- Application: the appropriate suspensions of the test substance was applied to three animals in volume 25 µl to the dorsum of each ear once a day morning for 3 consecutive days.
- Route of administration: the route of administration was the same as in the main study.
- Dose selection: the doses used in pilot experiment were chosen also for main study.

MAIN STUDY
Animal Check-in: animals 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.

Application of test substance
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 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 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 7.54 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed.

Examinations
- 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 signs were assessed using a defined scoring system described in internal SOP M/1 – twice daily during the dosing period. Efforts were made to characterize onset and duration of signs observed. All changes in behavior and health condition of animals will be recorded. Eg.: piloerection, ataxia, tremors, and convulsions, aggressiveness, change in grooming activity, marked change in activity level changes in frequency and intensity of breathing such as dyspnea, gasping, and rales etc.
- 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.
- Necropsy: the third day after last administration (five hours after application of radionuclide), all test animal were sacrificed by prolonged ether narcosis.

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 cm^2) 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 oC 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).

TREATMENT PREPARATION AND ADMINISTRATION
- Positive control concentration: a dose level for DNCB was determined during the verification of the method.
- Vehicle dosage: the vehicle and dosage volume were the same as in treated groups.
- Test solution: 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.

AUXILIARY MATERIALS
- Vehicle selection justification: the vehicle DAE 433 was selected according to reference (1). This vehicle is used in the testing laboratory for approximately 10 years and it elicited a consistent response over whole period.
- Chemicalos for preparation of cell suspension: PBS – Phosphate Buffered Saline, pH 7.4, Trichloroacetic acid 5 %, 3H-methyl thymidine.

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.

REFERENCE
(1) Ehling, G., Hecht, M., Heusener, A., Huesler, J., Gamer, A.O., van Loveren, H., Maurer, T., Riecke, K., Ullmann, L., Ulrich, P., Vandebriel, R. And Vohr, H.W. (2005), A European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round. Toxicol., 212, 60-68.

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB - 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.

Parameter:

SI

Value:

3.04

Test group / Remarks:

50 %

Remarks on result:

other: 3.04 at 50 %; 1.6 at 5 % and 0.98 at 0.5 %

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 672.00 at 50 %; 353.85 at 5 %; 216.30 at 0.5 %

Parameter:

SI

Value:

1.6

Test group / Remarks:

5 %

Parameter:

SI

Value:

0.98

Test group / Remarks:

0.5

MAIN STUDY

Summary of Results of Main Study

Group	Radioisotope incorporation		Ear weight
	Mean DPM	SI	Mean (mg)
NC	220.82	1.0	24.62
PC	1875.13*	8.49+	35.14*
50 %	672.00*	3.04+	26.84
5 %	352.85*	1.60	25.52
0.5 %	216.30	0.98	24.44

* = values statistically significant on probability level < 0.05 (Mann-Whitney test)

+ = values ≥ 3 

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

The animals exposed to the tested concentrations of the test substance showed no skin reactions and clinical symptoms of intoxication throughout the experiment.

The value of SI for the test groups treated by the test substance at the middle and lowest dose level is below the threshold, stimulation index (SI) is < 3 but the value of DPM at the middle dose level is statistically significantly increased compared to negative control.

 The value of SI for the highest dose level is ≥ 3 (50 % - 3.04) and the value of DPM is statistically significantly increased.

Statistically significant increase of ear weight and irritation to skin was not observed.

The comparison of the Stimulation Indexes and values of DPM between the treated groups and the control group revealed that the test substance caused a statistically significant increase in radioisotope incorporation into the DNA of dividing lymphocytes so the result of LLNA assay is positive.

Cell proliferation

The value of DPM and SI for positive control group was increased. The SI was ≥ 3 (8.49) – the LLNA was efficient.

The SI for the test groups treated by the test substance at the lowest and the middle dose level is below the threshold, stimulation index (SI) is < 3. The SI for the test group treated by the test substance at the highest dose level was ≥ 3 (50 % - 3.04).

The value of DPM at the highest and the middle dose levels is statistically significantly increased compared to negative control and dose-effect relationship is manifested.

Individual Activities and Calculated Values  

Group	NC	PC	50 %	5 %	0.50 %
(Anim.No.)	(1-5)	(6-10)	(11-15)	(16-20)	(21-25)
Activity (DPM)	204.65	1857.66	690.01	350.95	210.26
	190.55	1803.29	750.60	336.03	208.64
	203.12	1833.79	625.75	358.36	214.05
	224.33	1973.18	708.31	376.98	203.26
	281.44	1907.74	585.35	341.92	245.30
mean	220.82	1875.13*	672.00*	352.85*	216.30
SD	35.98	66.81	66.08	15.96	16.67
SI	1.00	8.49	3.04	1.60	0.98

NC: negative control; PC: positive control; SD: standard daviation

* = statistically significant on probability level < 0.05 (Mann-Whitney test)

Irritating Effect of the Test Substance

In the positive control group, the weight of ear target was statistically significantly increased against negative control group.

No erythema of skin was observed at all dose levels. Weights of ears were not markedly increased at all dose levels and values were relatively similar compared to the weight of the negative control group. Slight hint of dose-response relationship could be seen, but no statistical significance was at any of three tratment groups.

Individual ear weight

Group	NC	PC	50 %	5 %	0.50 %
(Anim.No.)	(1-5)	(6-10)	(11-15)	(16-20)	(21-25)
Weight of ear biopsies (milligrams)	24.3	38.0	25.2	27.0	24.6
	24.5	33.4	28.3	25.3	24.8
	24.2	30.1	29.9	26.5	23.8
	24.6	41.1	25.4	24.0	24.0
	25.5	33.1	25.4	24.8	25.0
mean	24.62	35.14*	26.84	25.52	24.44
median	24.50	33.4	25.4	25.3	24.6
SD	0.52	4.37	2.14	1.23	0.52

NC: negative control; PC: positive control; SD: standard daviation

* = statistically significant on probability level < 0.05 (Mann-Whitney test)

Body Weight of Animals

Individual body weight of females before administration and before necropsy was relatively well balanced (result of random selection of animals into groups). Very slight reduction of body weight (in tenths of grams) was recorded only in four animals at the highest and middle dose level. 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 treated group at the highest dose level. 

	Before application (1st day)					Before necropsy (6th day)
Group	NC	PC	50 %	5 %	0.50 %	NC	PC	50 %	5 %	0.50 %
(Anim.No.)	(1-5)	(6-10)	(11-15)	(16-20)	(21-25)	(1-5)	(6-10)	(11-15)	(16-20)	(21-25)
Body weight of animals (grams)	19.25	19.1	19.34	18	18.21	20.07	19.02	19.68	18.66	19.07
	18.53	18.26	18.97	18.72	18.56	19.32	19.19	18.26	18.95	19.34
	18.56	18.69	18.27	18.47	19.23	19.32	19.36	18.91	19.71	19.23
	18.03	18.74	19.42	19.21	18.92	18.9	19.73	19.15	19.2	20.02
	18.75	18.44	17.96	19.34	19.05	19.22	18.46	18.45	19.13	19.48
mean	18.62	18.65	18.79	18.75	18.79	19.37	19.15	18.89	19.13	19.42
SD	0.44	0.32	0.65	0.55	0.41	0.43	0.47	0.57	0.39	0.36

NC: negative control; PC: positive control; SD: standard daviation

Body Weight Increments

Group	NC	PC	50 %	5 %	0.50 %
(Anim.No.)	(1-5)	(6-10)	(11-15)	(16-20)	(21-25)
Body weight increment (grams)	0.82	-0.08	0.34	0.66	0.86
	0.79	0.93	-0.71	0.23	0.78
	0.76	0.67	0.64	1.24	0.00
	0.87	0.99	-0.27	-0.01	1.10
	0.47	0.02	0.49	-0.21	0.43
mean	0.74	0.51	0.10	0.38	0.63
SD	0.16	0.51	0.57	0.58	0.43

NC: negative control; PC: positive control; SD: standard daviation

Mortality, Clinical Observations

No animal died during the main study.

No symptoms of toxicity and no erythema on application site were 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 DNCB: hyperaemia of skin with well defined erythema on application site, clonospasm and increased response to stimuli.

Erythema - score

	Animal No.	Before application	1^stday	2^ndday	3^rdday	4^thday	5^thday	6^thday
NC	1	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0
	4	0	0	0	0	0	0	0
	5	0	0	0	0	0	0	0
PC	6	0	1	2	2	2	2	2
	7	0	1	2	2	2	2	2
	8	0	1	2	2	2	2	2
	9	0	1	2	2	2	2	2
	10	0	1	2	2	2	2	2
50 %	11	0	0	0	0	0	0	0
	12	0	0	0	0	0	0	0
	13	0	0	0	0	0	0	0
	14	0	0	0	0	0	0	0
	15	0	0	0	0	0	0	0
5 %	16	0	0	0	0	0	0	0
	17	0	0	0	0	0	0	0
	18	0	0	0	0	0	0	0
	19	0	0	0	0	0	0	0
	20	0	0	0	0	0	0	0
0.50 %	21	0	0	0	0	0	0	0
	22	0	0	0	0	0	0	0
	23	0	0	0	0	0	0	0
	24	0	0	0	0	0	0	0
	25	0	0	0	0	0	0	0

NC: negative control; PC: positive control

PILOT EXPERIMENT

Individual body weight of animals before administration was well balanced. Slight reduction of body weight after treatment was recorded in animal No. 2 (5 % w/v).

Animal No.	Before application	Day 6
1	18.61	18.74
2	18.41	18.21
3	18.59	18.58

During the pilot experiment no clinical symptoms of systemic toxicity were observed.

In treated animals no erythema and skin reaction were observed.

The thickness of ears in all animals during pilot experiment was without any changes.

During the pathological examination the auricular lymph nodes enlargement was not detected in all animals.

Mean Values of Thickness of Ears and Erythema Score in Pilot Experiment

	Animal No.	Mean thickness of right ear (mm)	Mean thickness of left ear (mm)	Erythema
Before application	1 (50 %)	0.19	0.20	0
	2 (5 %)	0.20	0.19	0
	3 (0.5 %)	0.19	0.20	0
1^stday	1	0.20	0.21	0
	2	0.20	0.19	0
	3	0.19	0.19	0
2^ndday	1	0.20	0.20	0
	2	0.20	0.19	0
	3	0.20	0.20	0
3^rdday	1	0.20	0.19	0
	2	0.20	0.20	0
	3	0.20	0.19	0
4^thday	1	0.19	0.20	0
	2	0.19	0.20	0
	3	0.20	0.20	0
5^thday	1	0.19	0.19	0
	2	0.20	0.19	0
	3	0.19	0.19	0

Interpretation of results:

other: skin sens 1B (H317), according to the CLP Regulation (EC 1272/2008)

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

May cause an allergicskin reaction.

Executive summary:

Method

The substance was tested for the assessment of skin sensitisation potential with the murine Local Lymph Node Assay. The Local Lymph Node Assay (LLNA) with radionuclide was used. The testing was conducted according to the OECD Test Guideline No. 429, Adopted 22nd July 2010.

The contact allergenic potential of the test item was evaluated after topical application to female BALB/c mice. Mice were exposed to three concentrations of test substance suspended in vehicle (DAE 433) 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.

Results

Comparison of Stimulation Indexes between treated groups and control vehicle group revealed that the test substance caused a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. The Stimulation Index of the highest treated groups is > 3 (50 % - 3.04). The value of DPM is statistically significantly increased compared to negative control. The Stimulation Index of the middle and the lowest treated groups (5 and 0.5 % w/v) is < 3 but the value of DPM at the middle dose level is statistically significantly increased compared to negative control and overall dose-effect relationship is manifested.

The test substance did not cause statistically significant increase of ear weight and irritation to skin at all dose level – it means the test substance did not cause irritation to skin. 

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

The positive control item DNCB (concentration 0.5 % (w/v) elicited a reaction pattern with statistically significant increase in Stimulation Index of cell proliferation and of ear weight, which was in congruence with his expected mode of action as a contact allergen.

Conclusion

According to the CLP Regulation (EC 1272/2008) a stimulation index of 3 or more is considered a positive response in the Local Lymph Node Assay and based on this type of test a substance in classified as: skin sensitiser sub-category 1A, if the EC3 values resulted to be equal/lower than 2 %; or skin sensitiser sub-category 1B, if the EC3 values resulted to be greater than 2 %.

The Stimulation Index of the highest treated groups is > 3 (3.04 - EC: 50 %), thus the test item can be classified as skin sensitiser, sub-category 1B (H317), according to the CLP Regulation (EC 1272/2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

No skin sensitisation data are available on Acid Brown 365 (ABr365).

The toxicity of ABr365 has been predicted on the basis of the structural conformation and on the basis of the related reactivity potential. ABr365 is a chromate-pyrazol sodium salt. Based on the structural and Qsar assessments, the mode of action as a contact allergen is attributable to the pyrazole moiety, which could play a special role in the sensitization reaction: if the oxygen is not involved in coordinate bonds, it may react with proteins, causing an sensitising effect.

Based on the possible mechanisms of action of the substance and based on the structural formula, the similar substance 1 seems to be the most similar and representative substance.

Similar substance 1 was tested for the assessment of skin sensitisation potential with the murine Local Lymph Node Assay, according to the OECD Test Guideline No. 429, Adopted 22nd July 2010. The contact allergenic potential of the test item was evaluated after topical application to female BALB/c mice. Mice were exposed to three concentrations of test substance suspended in vehicle (DAE 433) for 3 consecutive days.

The following concentrations in application forms were used: 50 %, 5 %, 0.5 % (w/v).

Comparison of Stimulation Indexes between treated groups and control vehicle group revealed that the test substance caused a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. The Stimulation Index of the highest treated groups is > 3 (50 % - 3.04). The value of DPM is statistically significantly increased compared to negative control. The Stimulation Index of the middle and the lowest treated groups (5 and 0.5 % w/v) is < 3 but the value of DPM at the middle dose level is statistically significantly increased compared to negative control and overall dose-effect relationship is manifested. The test substance did not cause statistically significant increase of ear weight and irritation to skin at all dose level – it means the test substance did not cause irritation to skin. The animals exposed to the test substance at all concentrations showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment.

The positive control item DNCB (concentration 0.5 % (w/v) elicited a reaction pattern with statistically significant increase in Stimulation Index of cell proliferation and of ear weight, which was in congruence with his expected mode of action as a contact allergen.

Migrated from Short description of key information:

Skin sensitiser

Justification for selection of skin sensitisation endpoint:

Study conducted on structural analogue, according to internationally accepted testing guidelines and performed according to GLP.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

The criteria to classify a substance as skin/respiratory sensitizer are reported into the second adaptation to technical progress*.

A stimulation index of 3 or more is considered a positive response in the Local Lymph Node Assay.Based on the Local Lymph Node Assay test results, a substance in classified as:

- skin sensitiser sub-category 1A, if the EC3 values resulted to be equal/lower than 2 %

- skin sensitiser sub-category 1B, if the EC3 values resulted to be greater than 2 %

Based on the read across approach, Acid Brown 365 (ABr365) can be considered as a skin sensiiser.

In conclusion, ABr365 is classified as skin sensitizer sub-category 1B (H317), according to the CLP Regulation (EC 1272/2008).

*Commission Regulation (EU) No 286/2011 of 10 March 2011, amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures