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Toxicological information

Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted on 17 July 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

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.

Study design: in vivo (non-LLNA)

Induction
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
Challenge
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)

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.