Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(3-)

Endpoint summary

Description of key information

Not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 20, 2018 to February 03, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted on 17 July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

According to the related guidelines, there are limitations applicable to the LLNA test in case of testing of certain metals.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Adita Biosys Private Limited, approved breeder.
- Females: females used were nulliparous and non-pregnant.
- Microbiological status of animals: veterinary examination of all animals was performed on the day of receipt and on 5th day of acclimation.
- Age at study initiation: 10 to 11 weeks.
- Weight at study initiation: 332.84 g to 349.36 g.
- Housing: single animal was housed in a standard polypropylene cage (size: 430 × 285 × 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet: altromin diet for Guinea pigs manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the animals throughout the experimental period.
- Water: water was provided ad libitum throughout the acclimation and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottle with stainless steel sipper tubes.
- Acclimation period: five days and thirteen days to experimental room conditions prior to treatment for pre study and main study, respectively.
- Indication of any skin lesions: animals were observed for clinical signs once daily.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.2 °C to 22.7 ºC
- Relative humidity: 42 % to 63 %
- Air changes: 12 to 15 air changes per hour.
- Photoperiod: 12 hours light and 12 hours dark cycle.

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

214 mg of test item was moistened with 214 μl of distilled water

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

214 mg of test item was moistened with 214 μl of distilled water

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Pre-study: 2 females
Main study: 10 females per vehicle control group; 20 females per test item group

Details on study design:

RANGE FINDING TESTS
- Site of exposure: pre-clipped area.
- Doses: 25, 50, 75 and 100 % w/v Test Item in vehicle (the concentration was correct by ingredient content).
- Dose selaction for the main test: the animals did not reveal any skin reactions on application of 214 mg of test item moistened with 214 μl of distilled water at 24 hours and 48 hours after patch removal in the pre-study. Hence, 214 mg of test item moistened with 214 μl of distilled water was selected for induction and challenge phase of the main study.
- Evaluation: skin reactions were be observed approximately at 24 and 48 hours post removal of the test patch. The skin reactions were recorded according to Draize method (1959

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 inductions at the day 0th, 7th and 14th.
- Frequency of applications: after 7 days.
- Test groups: 20 animals.
- Control group: 10 animals.
- Preparation of animals: approximately 24 hours before application, the fur from the left flank region and right flank region of the Guinea pigs was closely clipped using an electric hair clipper, exposing an area of approximately 80 sq. cm. Care was taken to avoid abrasion to the skin.
- Application: the test item was applied by occlusive patch on to the pre-clipped area of the skin and covered by approximately 4 cm × 6 cm cotton gauze patch.
- Duration: the test patch was held in its position for 6 hours, after which the test patches were removed and the area was cleaned with normal saline swabs and dried with cotton.
- Concentrations in test group: 214 mg of test item was moistened with 214 μl of distilled water (the concentration was correct by ingredient content);
- Concentration in control group: 214 μl of distilled water.
- Evaluation: the skin reactions were observed approximately at 1 and 24 hours post removal of the test patch after induction (topical application). The skin reactions were observed and recorded according to Draize method (1959).

B. CHALLENGE EXPOSURE
- No. of exposures: 1 challenge.
- Day of challenge: on the days 28th.
- Test groups: 20 animals.
- Control group: 10 animals.
- Preparation of animals: approximately 24 hours before application, the fur from the left flank region and right flank region of the Guinea pigs was closely clipped using an electric hair clipper, exposing an area of approximately 80 sq. cm. Care was taken to avoid abrasion to the skin.
- Application: the test item was applied by occlusive patch on to the pre-clipped area of the skin.
- Duration: the test patch was held in its position for 6 hours, after which the test patches were removed and the area was cleaned with normal saline swabs and dried with cotton.
- Concentrations in test group: 214 mg of test item was moistened with 214 μl of distilled water (the concentration was correct by ingredient content).
- Concentration in control group:214 μl of distilled water.
- Evaluation: the application sites were examined approximately at 24 hours (i.e., 30 hours after challenge application) and 48 hours (54 hours after the challenge application) after patch removal for skin reaction and scored according to Magnusson and Kligman grading scale.

ADDITIONAL OBSERVATIONS
- Clinical signs of toxicity and mortality: all the animals were observed once daily for clinical signs of toxicity and twice daily for mortality/morbidity during the experimental period.
- Body weight: individual animal body weight was recorded at receipt and on day 1 (before start of the treatment) and at termination for the pre study and main study animals.
- Necropsy: at the end of the observation period, all the animals were humanely sacrificed by carbon dioxide asphyxiation, subjected to necropsy and gross pathological examination and the observations were recorded.
- Pathology: no gross pathological changes were observed during the necropsy. Hence, histopathology was not carried out.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole (2-MBT)

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

214 mg of test item moistened with 214 μl of distilled water

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

214 μl of distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

75 % 2-MBT

No. with + reactions:

13

Total no. in group:

20

Clinical observations:

skin reactions like discrete or patchy erythema to no erythema

Remarks on result:

other: tested in a different study (no. BIO-ATX 219) From december 2018 to January 2019

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

75 % 2-MBT

No. with + reactions:

7

Total no. in group:

20

Clinical observations:

skin reactions like discrete or patchy erythema to no erythema

Remarks on result:

other: tested in a different study (no. BIO-ATX 219) From december 2018 to January 2019

No erythema and oedema were observed in both the vehicle control and treatment group animals in all the induction phases approximately at 1 and 24 hours observation after patch removal in main study.

No skin reactions were observed in both the vehicle control and treatment group animals in challenge phase approximately at 24 hours and 48 hours observations after patch removal in main study.

PRE-TEST

No erythema and oedema were observed in animals at any of the doses tested approximately at 24 and 48 hours after patch removal.

ADDITIONAL OBSERVATIONS

- Clinical signs of toxicity and mortality: no clinical signs of toxicity and mortality were observed in both the pre study and main study animals.

- Body weight: no treatment related change in body weight and percent change in body weight with respect to day 1 in any of the animals. All the animals showed physiologically normal increase in body weights in pre study and main study.

- Necropsy: no gross pathological changes were observed during the necropsy.

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Not skin sensitising

Executive summary:

The study was conducted to evaluate the skin sensitisation potential of test item in Guinea pigs by Buehler test method.

During the pre study, the dose of 53.5 mg and 107.0 mg of test item were applied topically to the anterior left flank and posterior left flank respectively, whereas 160.5 mg and 214.0 mg of test item were applied topically to the anterior right flank and posterior right flank respectively to each animal. No skin reactions were observed in any of the test dose approximately at 24 and 48 hours observation period after patch removal. No skin reactions were observed at the highest dose, hence, 214 mg of test item was selected for induction and challenge phase of main study.

The main study included three inductions on day 0, 7 and 14 and challenge on day 28. Induction day was performed with distilled water and 214 mg of test item moistened with 214 μl of distilled water. On challenge day 28, 214 μl of distilled water was topically applied to the anterior part of right flank, whereas 214 mg of test item moistened with 214 μl of distilled water was topically applied to the posterior part of the right flank of all the animals.

Durign the induction phase, no skin reactions were observed in vehicle control and treatment group animals approximately at 1 and 24 hours of observation period of post patch removal. During challenge phase, the animals did not reveal any skin reactions at anterior and posterior part of right flank of vehicle control and treatment group animals approximately at 24 and 48 hours of post patch removal.

No clinical signs of toxicity and mortality were observed till termination. All the treated animals revealed physiologically normal increase in body weights during the observation period. No gross pathological changes were observed in any of the animals.

Conclusion

Test item did not show any skin sensitisation or allergic potential in Guinea pigs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro studies were considered at first to assess the skin sensitisation potential of Acid Blue 193. In particular, key event 3 of skin sensitisation was assessed by h-CLAT (OECD 442E) that was found to be inconclusive, due to colour interference by the substance; a DPRA (OECD 442C) was not applicable to investigate key event 1 as the substance contains metal atoms; a study according to OECD 442D to explore key event 2 was not run, as not sufficient to draw a conclusion on the skin sensitising potential of the substance.

An in vivo LLNA (OECD 429) was not feasible as the substance contains metal atoms.

Consequently, a Bueheler assay (OECD 406) was run in Guinea pigs. It included 3 inductions on day 0, 7, 14 and a challenge on day 28. Test substance was applied topically in an amount of 214 mg in 214 µl. No skin reactions were seen in the induction phase; moreover, no signs of skin sensitisation were noted in tested animals 24 and 48 h after challenge.

No clinical signs of toxicity and mortality were observed till termination. All the treated animals revealed physiologically normal increase in body weights during the observation period. No gross pathological changes were observed in any of the animals.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Animal studies are used to classifiy for skin sensitisation within the CLP regulation (EC) No 1272/2008. Based on the Buehler assay, classification in category 1A applies for:

≥ 15 % responding at ≤ 0.2 % topical induction dose or

≥ 60 % responding at > 0.2 % to ≤ 20 % topical induction dose;

and classification in category 1B applies for:

≥ 15 % to < 60 % responding at > 0.2 % to ≤ 20 % topical induction dose or

≥ 15 % responding at > 20 % topical induction dose.

As no positive response was seen in tested animals, the substance was considered as devoid of a skin sensitising potential and was not classified.