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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 March 13 to April 27, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
1992
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
LLNA method is not adequate as the substance is a metal complex.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Adita Biosys Private Limited
- Age at receipt: 10 weeks
- Weight at study initiation: 320.47 to 341.42 g
- Housing: single animal was housed in a standard polypropylene cage (size: L 430 × B 285 × H 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: ad libitum
- Water: ad libitum
- Acclimation period: 5 days and 14 days to experimental room conditions prior to treatment for pre study and main study respectively; observation for clinical signs once daily; veterinary examination of all the animals on day of receipt and on day 5 of acclimatization.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.4°C to 22.8 ºC
- Humidity: 46 to 64 %
- Air changes: 12 - 15
- Photoperiod: 12 h light and 12 h dark cycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
100 mg of test item moistened with 0.1 ml of distilled water
Day(s)/duration:
day 0 / 6 h
Adequacy of induction:
other: highest concentration used in pre-study; it causes no skin reactions.
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
100 mg of test item moistened with 0.1 mL of distilled water
Day(s)/duration:
day 7 / 6 h
Adequacy of induction:
other: highest concentration used in pre-study; it causes no skin reactions.
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
100 mg of test item moistened with 0.1 ml of distilled water
Day(s)/duration:
day 14 / 6 h
Adequacy of induction:
other: highest concentration used in pre-study; it causes no skin reactions.
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
100 mg of test item moistened with 0.1 mL of distilled water
Day(s)/duration:
day 28 / 6 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
pre-study: 2
main study: 10 for vehicle control; 20 for test substance
Details on study design:
RANGE FINDING TESTS: doses of 25 %, 50 %, 75 % and 100 % w/v test item in vehicle were tested; no skin reactions were observed at any of test doses, hence highest dose of test item was selected for induction and challenge phase of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: day 0, day 7, day 14
- Test groups: 1
- Control group: 1
- Site: left and right flank
- Duration: 6 h
- Concentrations: 100 mg moistened in 0.1 ml of distilled water

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 28
- Exposure period: 6 h
- Test groups: 1
- Control group: 1
- Site: right flank
- Concentrations: 100 mg moistened in 0.1 ml
- Evaluation: 24 and 48 h after removal of the bandage

OTHER: skin reaction were scored according to Magnusson and Kligman grading scale.
The following observations were made during experimental period:
- 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.
- pathology
a. 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.
b. histopathology: no gross pathological changes were observed during the necropsy, thus histopathology was not carried out.
Challenge controls:
0.1 ml of distilled water
Positive control substance(s):
no
Remarks:
the reliability of the skin sensitisation was tested using 2-mercaptobenzothiazole in another study.

Results and discussion

Positive control results:
The positive control test was valid.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100 mg in 0.1 ml of distilled water
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100 mg in 0.1 ml of distilled water
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 mg in 0.1 ml of distilled water
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 mg in 0.1 ml of distilled water
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
75 mg moistened with 0.1 ml acetone
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
data from another study
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
75 mg moistened with 0.1 ml acetone
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
data from another study

Any other information on results incl. tables

Clinical signs of toxicity and mortality

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

Skin reactions scoring

No erythema and oedema were observed in G1 group animals at any of the doses tested approximately at 24 and 48 hours after patch removal in pre-study.

No erythema and oedema were observed in both the vehicle control (G2) and treatment group (G3) 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 (G2) and treatment group (G3) animals in challenge phase approximately at 24 hours and 48 hours observations after patch removal in main study.

     

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 in any of the animals in pre study and main study.

Applicant's summary and conclusion

Interpretation of results:
other: not classified within the CLP Regulation (EC 1272/2008)
Conclusions:
Not skin sensitiser.

Executive summary:

Method

Buehler test on Guinea pig according to OECD guideline 406.

In the pre-study, concentrations of 25 %, 50 %, 75 % and 100 % w/v were applied topically to the right flank of each animal. No skin reactions were observed in any of test concentrations approximately at 24 and 48 hours observation period after patch removal. As no skin reactions were seen, the highest concentration was selected for induction and challenge phase of main study.

The main study comprised a vehicle control group and a treatment group. Main study included 3 inductions on day 0, 7 and 14 and challenge on day 28.

On induction day 0, 7 and 14, 0.1 ml of distilled water and 100 mg of test item moistened with 0.1 ml of distilled water were applied topically on flank region. On challenge day 28, 0.1 ml of distilled as well as 100 mg of test item moistened with 0.1 ml of distilled water were topically applied on the flank of all the animals. Test patches were covered by approximately 4×6 cm2 cotton gauze and held in place with non-irritating adhesive tape and crepe bandage. After 6 hours of contact period, application sites were cleaned with normal saline swabs and dried. During induction phase, test item application sites were observed for skin reactions approximately at 1 and 24 hours post removal of test patches according to Draize method (1959) and during challenge phase the skin reactions were observed approximately at 24 and 48 hours post removal of the test patch according to Magnusson and Kligman grading scale.

All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality/morbidity during the experimental period. 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.

Results

In 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.

In challenge phase, animals did not reveal any skin reactions in both vehicle control and treatment groups approximately at 24 and 48 hours of post patch removal.

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

No gross pathological changes were seen at necropsy in any of the animals.