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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
other: read across from analogue substance
Adequacy of study:
key study
Study period:
From Oct. to Nov. 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Deviations:
yes
Remarks:
scale for skin reactions differs from protocol; 10 control animals used instead of 20; test animal age not specified
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study (different from LLNA test) is available.

Test material

Constituent 1
Reference substance name:
-
EC Number:
400-320-4
EC Name:
-
Cas Number:
94933-05-8
Molecular formula:
not applicable
IUPAC Name:
Reaction products of diazotised anthranilic acid coupled with resorcinol and diazotised 5-aminonaphthalene-1-sulphonic acid, metallised with Chromium (III) basic sulphate, sodium salts
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Hartley-Dunkin
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hacking and Churchill Ltd., Abbots Ripton Road, Wyton, Huntingdon, PE17 2DT, England
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals: not specified
- Age at study initiation: not specified
- Weight at study initiation: 291 to 328 g
- Housing: groups of one or two guinea pigs by dose group, in grid floor polypropylene cages
- Diet: ad libitum; Guinea Pig Diet, Standard with Vitamin C (Special Diets Services Ltd., Stepfield, Witham, Essex, England)
- Water: ad libitum ; certificate to demonstrate the quality of the drinking water of Yorkshire Water Authorityn provided
- Acclimation period: 5 days
- Indication of any skin lesions: not specified
- Randomisation: no formal randomisation procedure; transferred upon arrival in order of left to right
- Identification: indelible ink markings on the ears (Inscribe 700, Inscribe Ltd., Alton, Hants, England)

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 23 °C
- Humidity: 49 to 74 %
- Air changes: not specified
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Remarks:
distilled
Concentration / amount:
5.0 % (w/v)
Day(s)/duration:
one pair of injections
Route:
intradermal
Vehicle:
other: Complete adjuvant 50:50 v/v in water
Concentration / amount:
0 % (w/v)
Day(s)/duration:
one pair of injections
Route:
intradermal
Vehicle:
other: Complete adjuvant 50:50 v/v in water
Concentration / amount:
5.0 % (w/v)
Day(s)/duration:
one pair of injections
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
distilled
Concentration / amount:
25 % (w/v)
Day(s)/duration:
48 hours
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
distilled
Concentration / amount:
25 % (w/v)
Day(s)/duration:
24 hours
No. of animals per dose:
Test group: 20 guinea pigs (10 male and 10 female)
Control group: 10 guinea pigs (5 male and 5 female)
Details on study design:
RANGE FINDING TESTS: previous experience with the test item indicated that it was non-irritant by topical application. No screening study was considered necessary and concentrations of 5.0 % w/v in distilled water and adjuvant and 25 % w/v in distilled water were selected for the intradermal injections and the topical induction and challenge applications.

MAIN STUDY

A. INDUCTION EXPOSURE

I) Induction by intradermal injection: 3 pairs of 1.0 ml injections were administered to the clipped shoulder region of 20 test animals and 10 control animals using 26 gauge sterile hypodermic needles attached to a 1 ml disposable plastic syringe.
- No. of exposures: 3 pairs of 1.0 ml injections
- Test groups: 20 guinea pigs (10 male and 10 female)
- Control group: 10 guinea pigs (5 male and 5 female)
- Site: 4 x 6 cm clipped area on the shoulder region
- Frequency of applications: singluar injections
- Test concentrations: 1st pair: 5.0 % test item in distilled water; 2nd pair: complete adjuvant (50 % v/v in water); 3rd pair: 5.0 % test item emulsified in 50 % v/v complete adjuvant
- Control concentrations: 1st pair: distilled water; 2nd pair: complete adjuvant (50 % v/v in water); 3rd pair: complete adjuvant (50 % v/v in water)
- Duration: day 1

II) Induction by topical application: one week after the induction by intradermal injection, the same area was re-clipped and a 2 x 4 cm patch of Whatman no. 3 mm filter paper saturated in 25 % w/v test item was applied to the area, and secured with overlapping occlusive tape for 48 hours (control group administered vehicle only).
- No. of exposures: 1
- Exposure period: 48 hours
- Test groups: 20 guinea pigs (10 male and 10 female)
- Control group: 10 guinea pigs (5 male and 5 female)
- Site: 4 x 6 cm clipped area on the shoulder region
- Frequency of applications: once
- Concentration: 25 % (test group)
- Duration: days 8 to 10

B. CHALLENGE EXPOSURE
Challenge by topical application: a 5 x 5 cm area was clipped on both the right and left flanks of test and control animals. A 2 x 2 cm filter paper pad saturated in 25 % w/v test item in distilled water was applied to the left flank of all animals and secured with overlapping occlusive tape for 24 hours. A similar pad saturated in distilled water was applied similarly to the right flank of all animals.
- No. of exposures: 1
- Duration: two weeks after induction by topical application
- Exposure period: 24 hours
- Test groups: 20 guinea pigs (10 male and 10 female)
- Control group: 10 guinea pigs (5 male and 5 female)
- Site: left and right flanks
- Concentrations: 25 % w/v in distilled water and 0 % in distilled water (all animals)
- Evaluation: 24 and 48 hours after the removal of the dressing
Challenge controls:
Induction by injection: vehicle only
Induction by topical application: treated the same as test animals
Challenge: treated the same as test animals
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Test challenge (25 % w/v test item in distilled water; left flank)
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Test challenge (25 % w/v test item in distilled water; left flank)
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Vehicle challenge (0 % test item in distilled water; right flank)
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Vehicle challenge (0 % test item in distilled water; right flank)
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Test challenge (25 % w/v test item in distilled water; left flank)
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
Test challenge (25 % w/v test item in distilled water; left flank)
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Vehicle challenge (0 % test item in distilled water; right flank)
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
Vehicle challenge (0 % test item in distilled water; right flank)
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

Table 1: Results summary.

Observation time Number showing response at challenge (%) Classification
Test Control
L R L R
24 0/20(0) 0/20(0) 0/10(0) 0/10(0) non-sensitiser
48 0/20(0) 0/20(0) 0/10(0) 0/10(0)

L = left flank - test challenge site

R = right flank = vehicle challenge site

Table 2: Skin reactions in test animals following challenge application of the test item.

Animal number  Gender Weight (g) Skin reactions 
24 48
L R L R
1 M 315 0 0 0 0
2 313 0 0 0 0
3 311 0 0 0 0
4 315 0 0 0 0
5 318 0 0 0 0
6 292 0 0 0 0
7 304 0 0 0 0
8 291 0 0 0 0
9 292 0 0 0 0
10 296 0 0 0 0
11 F 315 0 0 0 0
12 313 0 0 0 0
13 311 0 0 0 0
14 322 0 0 0 0
15 311 0 0 0 0
16 324 0 0 0 0
17 308 0 0 0 0
18 307 0 0 0 0
19 307 0 0 0 0
20 293 0 0 0 0

L = left flank - test challenge site

R = right flank = vehicle challenge site

Table 3: Skin reactions in control animals following challenge application of the test item.

Animal number  Gender Weight (g) Skin reactions 
24 48
L R L R
21 M 291 0 0 0 0
22 311 0 0 0 0
23 328 0 0 0 0
24 310 0 0 0 0
25 300 0 0 0 0
26 F 305 0 0 0 0
27 312 0 0 0 0
28 315 0 0 0 0
29 318 0 0 0 0
30 295 0 0 0 0

L = left flank - test challenge site

R = right flank = vehicle challenge site

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP Regulation (EC 1272/2008)
Conclusions:
The substance was not found to be a sensitiser under the test conditions.
Executive summary:

Skin sensitisation potential was evaluated in an experimental study according to the Guinea Pig Maximisation Test (GPMT), comparable to the OECD Guideline 406 (1981). A separate, preliminary study found that the test item was non-irritant to skin, so the following test concentrations were determined as appropriate: 5.0 % (w/v) in distilled water and in adjuvant for induction by intradermal injection; and 25 % (w/v) in distilled water for induction by topical induction and for the challenge applications.

Induction: 10 male and 10 female guinea pigs were administered 3 pairs of 0.1 ml intradermal injections along the side of a 4 x 6 cm area on the clipped shoulder region. The three pairs of injections contained the following: 1. 0.1 ml 5.0 % (w/v) test item in distilled water; 2. 0.1 ml Complete adjuvant (50 % v/v in water); 3. 0.1 ml 5.0 % (w/v) test item emulsified in 50 % (v/v) Complete adjuvant. An additional 5 male and 5 female control guinea pigs were administered three pairs of injections containing: 1. 0.1 ml distilled water; 2. 0.1 ml Complete Adjuvant (50 % (v/v) in water); 3. 0.1 ml Complete Adjuvant (50 % (v/v) in water. One week later, the same region was re-clipped and a 2 x 4 cm patch of Whatman No. 3 filter paper, saturated in 25 % (w/v) test item (test group) or vehicle (control group), was secured using overlapping occlusive tape for 48 hours.

Challenge: Two weeks later, the flanks of test and control animals were clipped (5 x 5 cm area). 2 x 2 cm patch of filter paper saturated with 25 % test item was applied to the left flank, and another saturated with distilled water to the right flank, with overlapping occlusive tape for 24 hours, at which time the dressing was removed. Skin reactions were recorded 24 and 48 hours after the dressing was removed.

No skin reactions were recorded in either test or control groups during the study period, at either 24 or 48 hours after treatment.

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