Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-01-25 to 1999-02-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study reliable with restrictions Deviations from EU method B.6 (2008) and OECD 406 (1992): - the positive control study was conducted in 1997, but this study is from 1999. According to the guidelines a postive control study should be conducted every six months. - the age of the animals was not stated - the justification for the choice of the vehicles is missing in the study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1996
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. defiencies" above
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992-07-17
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. defiencies" above
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- CAS name: 1H-Benzimidazole-4,6-disulfonic acid-2,2´-(1,4-diphenylene) bis, disodium salt
- Molecular formula: C20H12N4O12S4Na2
- Molecular weight: 674g/mol
- physical state: solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Tierzucht Schönwalde GmbH, D-16352 Schönwalde
- Weight at study initiation: between 318 and 397 g
- Housing: the guinea pigs wwere housed in polycarbonate 8macrolone type IV, floor area 1800 cm^2) cages, two or three to a cage. The bedding used was softwood sawdust "Lignocel-Fasern" from Altromin, D-32791 Lage, Lippe.
- Diet (ad libitum): a pelleted diet "Altromin 3122", from Altromin, D-32791 Lage, Lippe
- Water (ad libitum): vitamin C enriched domestic quality water acidified to pH 2.5 with hydrochloric acid in order to prevent microbiological growth
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21°C ± 3°C
- Relative humidity: 55% ± 15%
- Air changes: 10 times/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: sterile distilled water and acid free vaseline (DAB 10)
Concentration / amount:
Intradermal injection: 0.5% (w/w) test article concentration in sterile distilled water
Dermal induction: 25% (w/w) test article concentration in acid free vaseline (DAB 10)
Challenge application: 10% (w/w) test article concentration in acid free vaseline (DAB 10)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: sterile distilled water and acid free vaseline (DAB 10)
Concentration / amount:
Intradermal injection: 0.5% (w/w) test article concentration in sterile distilled water
Dermal induction: 25% (w/w) test article concentration in acid free vaseline (DAB 10)
Challenge application: 10% (w/w) test article concentration in acid free vaseline (DAB 10)
No. of animals per dose:
Preliminary test: 10 animals (2 animals for intradermal injection; 4 animals for topical application; 4 animals for topical application with Freund' Complete Adjuvans: Aqua dest. = 1:1 applicated intradermally)
Main study: 10 animals (test group) plus 5 animals (control group)
Positive control group: not stated
Details on study design:
RANGE FINDING TESTS:
The intradermal irritancy of the test article was investigated in order to find the minimal irritant test article concentration for the intradermal induction using intradermal injections as in the main study. Concentrations used were as follows: 0.5%, 1%, 2% and 5%.
The topical irritancy of the test article was investigated in order to find the minimal irritant test article concentration for the dermal induction, and the maximum non-irritating test article concentration for the challenge application using the same procedure like in the main study in 4 animals. Concentration used were as follows: 5%, 10%, 25% and 40%. A group of 4 other animals were treated intradermally with Freund' Complete Adjuvans: Aqua dest. = 1:1 three days before dermal application. Concentration used were as follows: 5%, 10%, 25% and 40%.

Results:
- Intradermal irritancy: moderate and confluent erythema (grade 2) was observed at the 1%, 2% and 5% concentrations during the 24 and 48 hour observations. At the 0.5% concentration moderate and confluent erythema (grade 2) was observed in one animal at the 24 and 48 hour observation, while moderate and confluent erytehma (grade 2) was observed in one animal at the 24 hour observation and slight or discrete erythema (grade 1) was observed in the same animal at the 48 hour observation.
- Topical irritancy (not FCA treated): no skin reactions were observed at any concentration tested, except for slight or discrete erythema (grade 1) in one animal at the 48 hour osbervation.
- Topical irritancy (FCA treated): slight or discrete erythema (grade 1) was observed at the 25% and 40% concentration at the 48 hour observation. The same type of erythema was observed at the 5% and 10% concentration in one animal, respectivley at the 48 hour observation.
On the basis of the results the following dose levels were selected for the main study: a 0.5% (w/w) test article concentration was selected for the intradermal induction, whereas a 25% (w/w) test article concentration was chosen for the dermal induction and a 10% (w/w) test article concentration was used for the challenge application.

MAIN STUDY
A. INDUCTION EXPOSURE
Day -1:
An area of dorsal skin 4 x 6 cm in the scapular region was clipped free of hair with an electric clipper.
Day 0:
Three pairs of intradermal injections were given simultaneously into this area. Each dose was 0.1 ml.
The first two pairs of injections were given close to each other and most cranially, while the third pair of injections was given towards the caudal part of the test area.
- 1st pair: Freund's complete adjuvant (FCA) diluted 1:1 with sterile water
- 2nd pair: control group: sterile distilled water; test group: 0.5% (w/w) test article in sterile distilled water
- 3rd pair: control group: equal amounts of sterile distilled water and FCA/sterile distilled water in the ratio 1:1 (w/w); test group: equal amounts of 1% (w/w) test article in sterile distilled water and FCA/sterile distilled water in the ratio 1:1 (w/w)
Day 6:
Six days after the injections, the same 4 X 6 cm scapular area was clipped free of hair and assessed for irritancy.
Day 7:
A paper patch (Whatman No. 3M, 2 X 4 cm) was saturated with about 0.25 g of the vehicle (control group) or a 25% test article concentration (test group). the patch was placed on the skin and covered with impermeable adhesive bandage (Blenderm 5 X 7 cm). this was firmly secured with tape (Gothaplast 5 cm width) wrapped round the trunk. The dressing was left in place for 48 hours.
Day 9:
The test area was assessed for irritancy after removing the bandage.

B. CHALLENGE EXPOSURE
Day 20:
Hair was removed with an electric clipper and an electric razor from a 4 X 6 cm area from the left flank of the guinea pigs.

Day 21:
A paper patch (Whatman No. 3M, 2 X 2 cm) was saturated with about 0.1 g of 10% (w/w) test article and another one with the vehicle and placed on the left anterior and left posterior flank of the guinea pigs, respectively.
The patches were covered with impermeable adhesive bandage (Blenderm 4 x 5 cm width) and secured with tape (Gothaplast 5 cm width) wrapped round the trunk. After 24 hours of exposure the patches and tape were removed.
Day 23 and 24
Each test area was examined 24 and 48 hours after removal of the patch. About three hours before every reading the sites were clipped and shaved in order to facilitate the evaluation.
Rections were scored according to the Magnusson and Kligman scale.

OBSERVATIONS:
- Health and body weight: the animals were observed at least once a day for signs of ill health and reaction to treatment.
The animals were weighed on the day of the first induction (day 0) and at termination of the study (day 24).
Challenge controls:
Vehicle control group:sterile distilled water and acid free vaseline (DAB 10)
5 females were used for the vehicle control group
Positive control substance(s):
yes
Remarks:
hexylcinnamaldehyde (from Aldrich, USA; 10% in sesame oil for intradermal induction and undiluted for topical induction and challenge)

Results and discussion

Positive control results:
60% of the animals responded positively.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10% concentration of the test item
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Slight erythema (grade 1); the slight reaction seen in the animal 24 hours after treatment as well as with the vehicle were due to other factors than sensitisation. It might be an irritation effect.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% concentration of the test item. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Slight erythema (grade 1); the slight reaction seen in the animal 24 hours after treatment as well as with the vehicle were due to other factors than sensitisation. It might be an irritation effect..
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10% concentration of the test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reactions were observed in any animal after 48 hours.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% concentration of the test item. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions were observed in any animal after 48 hours..
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
acid free vaseline (DAB 10)
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
slight erythema (grade 1); this is same animal, which showed a reaction to the test item; please refer to the results of the treatment with the test item
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: acid free vaseline (DAB 10). No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: slight erythema (grade 1); this is same animal, which showed a reaction to the test item; please refer to the results of the treatment with the test item.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
acid free vaseline (DAB 10)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reactions were observed in any animal after 48 hours.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: acid free vaseline (DAB 10). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions were observed in any animal after 48 hours..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10% concentration of the tet item
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reactions were observed in any animal.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10% concentration of the tet item. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were observed in any animal..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10% concentration of the test item
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reactions were observed in any animal.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10% concentration of the test item. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were observed in any animal..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
acid free vaseline (DAB 10)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reactions were observed in any animal.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: acid free vaseline (DAB 10). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were observed in any animal..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
acid free vaseline (DAB 10)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reactions were observed in any animal.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: acid free vaseline (DAB 10). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions were observed in any animal..

Any other information on results incl. tables

Induction of test and control group

Intradermal injections of Freund's omplete adjuvant mixed with vehicle or test article elicited irritation. No skin reactions were observed following the dermal induction with neither vehicle nor test article.

Observations

- health and body weight: the animals showed no signs of ill health. The animals had normal body weight gain during the study period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The slight reactions seen in one out of 10 animals of the test group 24 hours after treatment with the 10% test article as well as with the vehicle were due to other factors than sensitisation. It might be an irritation effect.
All other animals of the control group as well as of the teet group were free of skin reactions after treatment with the test article and with the vehicle.
It was concluded that the test substance was not generative of provoking a delayed contact hypersnesitivity in guinea pigs.
According to 67/548/EC and subsequent regulations, the substance is not classified as a skin sensitiser.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the substance is not classified as a skin sensitiser.