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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 15 APRIL 1997 to 15 APRIL 1997

The first induction was performed on 15.04.1997 and the last examination of the skin was performed on 15.05.1997.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study in accordance with guidelines by OECD, "Skin sensitization", No. 406, 1992, and the EEC guideline EEC 92/69 part B6, 1992

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Method recommended in the OECD Guideline No. 406, "Skin Sensitization", 1992 and the EEC Guideline "EEC 92/69 part B6", 1992. The test used was the Buehler test (Arch. Derm. 91, 171-177, 1965).
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
There was no legislative requirement to use the LLNA method at the time the study was conducted.

Test material

Constituent 1
Reference substance name:
Ichthyolic acid, sodium salt
EC Number:
215-671-7
EC Name:
Ichthyolic acid, sodium salt
Cas Number:
1340-06-3
IUPAC Name:
Identification of the UVCB substance by "Chemcial Abstracts Index Name", among others: Ichthyolic Acid, Sodium Salt. No IUPAC name known.
Test material form:
liquid
Specific details on test material used for the study:
For the study test material from regular production at the registrant's manufacturing site was used (batch R960045).

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Animals
Thirty female SPF albino guinea pigs of the Dunkin Hartley stock were obtained from Charles River GmbH, D-97633 Sulzfeld. At start of the experiment the guinea pigs weighed between 302 and 377 g. The experiment was preceded by an acclimatization period of 6 days. For the preliminary investigations another 4 animals were used.

Housing
The study took place in animal room No. 6 provided with filtered air at a temperature of 21 °C ± 3°C, relative humidity of 55% ± 15% and air change 10 times/hour. Humidity was higher for a short period during the study. This was assumed not to influence the outcome of the study. The room was illuminated to give a 24 hour cycle of 12 hours light and 12 hours darkness. Light was on from 06 to 18 h.
The guinea pigs were housed in polycarbonate (macrolone type IV, floor area 1800 cm2) cages, two or three to a cage. The cages were cleaned and the bedding changed 3 times a week.

Bedding
The bedding used was softwood sawdust "Lignocel-Fasern" from Altromin, D-32791 Lage, Lippe. Analyses for possible contaminants are performed regularly. Certificates of analysis of bedding are retained.

Diet
The guinea pigs had free access to a pelleted diet "Altromin 3122", from Altromin, D-32791 Lage, Lippe. Analyses for major nutritive components and significant contaminants are performed regularly. Certificates of analysis are retained.

Drinking water
The guinea pigs had free access to vitamin C enriched domestic quality water acidified to pH 2.5 with Hydrochloric acid in order to prevent microbiological growth. Analyses for possible contaminants are performed regularly. Certificates of analysis are retained.

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
The induction phase in the "test group" consisted of 3 weeks with application of a closed patch for 6 h each week. In the first 2 weeks the test substance was applied undiluted and in the 3rd week it was applied in a concentration of 75% (in water).
Concentration / amount:
test group
1st and 2nd induction: Ichthyol®, pale, undiluted
3rd induction: Ichthyol®, pale, 75% (w/w) in sterile distilled water

control group
use of sterile distilled water for the whole induction phase of three weeks with application of a closed patch for 6 h/w.
Day(s)/duration:
(day 0) 6h closed patch
(day 7) 6 h closed patch
(day 14) 6 h closed patch
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#20
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
The following dose levels were selected: For dermal inductions the test article was used undiluted. For the third induction the test article concentration was decreased to 75% (w/w), because moderate to marked skin irritation occured during the undiluted application. It was decided to repeat the preliminary investigation in order to find a non-irritating test article concentration for the challenge application. Consequently a 25% (w/w) test article concentration was chosen for challenge application.
For "challenge" an area (4x6 cm) of skin in the right flank region was clipped free of hair with an electric razor. A paper patch (Whatman No. 3M, 2.5 x 2.5 cm) was saturated with about 0.2 ml of the test article in a 25% concentration.
The patch was placed on the skin of the anterior part of the right flank. In the same way was a paper patch (Whatman No. 3M, 2.5 x 2.5 cm) saturated with about 0.2 ml of the vehicle and placed posterior on the right flank. The patches were fixed with impermeable tape (Blenderm, 5x5 cm). The trunk of the animal was wrapped with Gothaplast tape (5 cm width). The dressings were removed after 6 hours of exposure.
Day(s)/duration:
Induction
(day 0) 6 h closed patch undiluted test substance
(day 7) 6 h closed patch undiluted test substance
(day 14) 6 h closed patch 75% test substance in water

Challenge
(day 28) 6 h closed patch test 25% test substance in water
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 (controls)
20 (test group)
Details on study design:
Animal identification and allocation to groups
The guinea pigs were randomly allocated into 2 groups and earmarked with a punched number. To ensure that each animal could easily be identified the guinea pig with the highest number in a cage was marked on the forehead with a red India ink pen and the guinea pig with the second highest number was marked green.

Group 1 animal number 1 - 10 treatment sterile distilled water
Group 2 animal number 11 - 30 treatment ICHTHYOL PALE

Dose levels
On the basis of the preliminary investigation described below, the following dose levels were selected: For dermal inductions the test article was used undiluted. For the third induction the test article concentration was decreased to 75% (w/w), because moderate to marked skin irritation occured during the undiluted application. It was decided to repeat the preliminary investigation in order to find a non-irritating test article concentration for the challenge application. Consequently a 25% (w/w) test article concentration was chosen for challenge application.

Testing procedure
Preliminary irritation investigations
With another four animals, suitable concentrations of the test article were assessed for irritancy for the topical applications of the induction and challenge phases.
The procedures used in the preliminary investigation were identical to those used in the main study. The results are presented in Appendix I.

Induction
On the first experimental day (day 0), an area of skin in the left flank region measuring 4 x 6 cm was clipped free of hair with an electric clipper and an electric razor. A paper patch (Whatman No. 3M, 2.5 x 2.5 cm) was saturated with about 0.2 ml of the undiluted test article. The patch was placed on the skin of all the animals in the test group and fixed with impermeable tape (Blenderm, 5x5 cm). The trunk of the animal was wrapped with Gothaplast tape (5 cm width). The dressings were removed after 6 hours.
The procedure was repeated on day 7 with the undiluted test article and on day 14 with 75% test article concentration.
The test sites were assessed for irritancy 24 hours after each patch application.
The guinea pigs in the control group were treated in a similar manner, with the vehicle only.

Challenge
Four weeks after the first induction (day 28) all test and ^control group' animals were challenged.
An area (4x6 cm) of skin in the right flank region was clipped free of hair with an electric razor. A paper patch (Whatman No. 3M, 2.5 x 2.5 cm) was saturated with about 0.2 ml of the test article in a 25% concentration.
The patch was placed on the skin of the anterior part of the right flank. In the same way was a paper patch (Whatman No. 3M, 2.5 x 2.5 cm) saturated with about 0.2 ml of the vehicle and placed posterior on the right flank. The patches were fixed with impermeable tape (Blenderm, 5x5 cm). The trunk of the animal was wrapped with Gothaplast tape (5 cm width). The dressings were removed after 6 hours of exposure.


Positive control substance(s):
yes

Results and discussion

Positive control results:
At regular intervals the animal strain used, the sensitivity and reliability of the experimental technique was assessed using 2-mercaptobenzothiazole from Merck, Germany (50% for induction and challenge) dissolved in sesame oil. The latest positive control study was performed from 24.07.1995 to 25.08.1995 in which 17% of the animals responded positively.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.2 ml in 25% concentration
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None of the animals showed signs of ill health.
Remarks on result:
no indication of skin sensitisation
Remarks:
Slight erythema was observed in three animals (animal Nos. 14, 16 and 20) after 24 hours. After 48 hours no skin reactions were recorded in these animals. A score 1 is considered a marginal skin change due to other factors than sensitization.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.2 ml of the test article in a 25% aqueous solution
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None of the animals showed signs of ill health.
Remarks on result:
no indication of skin sensitisation
Remarks:
Slight erythema was observed in three animals (animal Nos. 14, 16 and 20) after 24 hours. After 48 hours no skin reactions were recorded in these animals. A score 1 is considered a marginal skin change due to other factors than sensitization.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.2 ml of the test article in a 25% aqueous solution
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reactions were recorded at any of the examinations. None of the animals showed signs of ill health.
Remarks on result:
no indication of skin sensitisation
Remarks:
In the negative control group ICHTHYOL PALE was used in "challenge" on the right anterior flank of the animals in a 25% aqueous solution (in addition to use of water on the right posterior flank). No skin reactions were recorded at any of the examinations.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.2 ml of the test article in a 25% aqueous solution
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reactions were recorded at any of the examinations. None of the animals showed signs of ill health.
Remarks on result:
no indication of skin sensitisation
Remarks:
In the negative control group ICHTHYOL PALE was used in "challenge" on the right anterior flank of the animals in a 25% aqueous solution (in addition to use of water on the right posterior flank). No skin reactions were recorded at any of the examinations.
Key result
Reading:
other: Reliability check
Group:
positive control
Dose level:
2-mercaptobenzothiazole from Merck, Germany (50% for induction and challenge) dissolved in sesame oil
Remarks on result:
positive indication of skin sensitisation
Remarks:
With regard to a positive control group following information is given in the study report: At regular intervals the animal strain used, the sensitivity and reliability of the experimental technique is assessed using 2-mercaptobenzothiazole from Merck, Germany (50% for induction and challenge) dissolved in sesame oil. The latest positive control study was performed from 24.07.1995 to 25.08.1995 in which 17% of the animals responded positively.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Executive summary:

The dermal sensitizing potential of ICHTHYOL PALE was investigated according to one of the methods recommended in the OECD Guideline No. 406, "Skin Sensitiziation", 1992 and the EEC Guideline "EEC92/69 part B6", 1992. The test used was the Buehler test (Arch. Derm. 91, 171-177, 1965).

The experiment was performed on 30 guinea pigs divided into a test group of 20 animals, and a control group of 10 animals. The study included an induction and a challenge phase. The animals in the test group were induced with the test article and the animals in the control group were induced with distilled water. The induction procedure included a closed patch topical application for 6 hours once a week for 3 weeks.

The challenge procedure included a closed patch topical treatment of the test article on the flank 4 weeks after the first induction. All animals were challenged for 6 hours. The skin reactions were evaluated 24 and 48 hours after termination of the challenge application.

Undiluted test article was used for the first two inductions and a concentration of 75%(w/w) in distilled water was used for the third induction. A 25% (w/w) test article concentration in distilled water was used for the challenge.

Under experimental conditions described in this report ICHTHYOL PALE caused no evidence of delayed contact hypersensitivity.