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Administrative data

Description of key information

The substance was found to be a skin sensitizer of high potency in the Guinea Pig Maximization test (OECD 406).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
yes
Remarks:
two challenge concentrations tested on one animal.
Principles of method if other than guideline:
Purity: no data
- Name in report: Cheque Dye A/S
- Physical state: black powder
- Lot/batch No.: 01-0118
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed prior to the establishment of the LLNA.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 337 - 422g
- Housing: groups of up to ten in suspended metal cages with wire mesh floors
- Diet (e.g. ad libitum): guinea-pig diet FD2 enriched with vitamin C, ad libitum; hay was additionally given weekly
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs


Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Induction, intradermal injection: 0.5% w/v in water
Induction, topical application: 10% w/v in water
Challenge, topical application: 7.5 and 4% w/v in water
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction, intradermal injection: 0.5% w/v in water
Induction, topical application: 10% w/v in water
Challenge, topical application: 7.5 and 4% w/v in water
No. of animals per dose:
Control animals: 10/group
Test animals: 25/group
Details on study design:
RANGE FINDING TESTS
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.


MAIN STUDY
A. INDUCTION EXPOSURE
Induction intradermal injections
The control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injection.
A 40 x 60 mm area of dorsal skin on the scapular region of each test animal was clipped free of hair. Three pairs of intradermal injections were made into a 20 x 40 mm area within the clipped area:
- Freund's complete adjuvant diluted 1:1 with water
- Test material 0.5%, w/v in water
- Test material 0.5% w/v in a 1:1 mixture of Freund's complete adjuvant and water

Induction topical application
The control animals were treated similarly to the test animals with the exception that the test substance was omitted from the topical application.
One week after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair. After 24 hours, a 20 x 40 mm patch saturated with ca. 0.4 mL of 10% test material in water was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape. This in turn was firmly secured by elastic adhesive bandage wound round the torso of the animal and fixed with impervious plastic adhesive tape. The dressing was left in place for 48 hours.

B. CHALLENGE EXPOSURE
The control and test animals were challenged topically two weeks after the topical induction application; the test concentrations were 7.5 and 5% w/v in water. Hair was removed by clipping and then shaving from an area on the left flank of each animal. A 20 x 20 mm patch was saturated with approximately 0.2 mL of 7.5% test material in water and applied to an anterior site on the flank. The test material at a concentration of 4% in water was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours and the dressing was secured as described for the topical induction.

EXAMINATIONS
The dermal reactions were assessed using the following numerical system:
Erythema and eschar formation:
No erythema 0
Slight erythema 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Edema formation:
No edema 0
Slight edema 1
Well-defined edema (edges of area well-defined by definite raising) 2
Moderate edema (raised approximately 1 mm) 3
Severe edema (raised more than 1 mm and extending beyond the area of exposure) 4

INTERPRETATION OF RESULTS
Dermal reactions in the test animals elicited by the challenge application were compared with the findings simultaneously obtained in the control animals.
A test animal was considered to show positive evidence of delayed contact hypersensitivity if the observed dermal reaction at challenge was definitely more marked and/or persistent than the maximum reaction seen in animals of the control group.
If the dermal reaction seen in a test animal at challenge was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in control animals, the result for that test animal was classified as inconclusive.
A test animal was considered to show no evidence of delayed contact hypersensitivity if the dermal reaction resulting from the challenge application was the same as, or less marked and/or persistent than the maximum reaction seen in animals of the control group.

Moreover, all animals were observed daily for signs of ill health or toxicity, and body weight gain was assessed.
Positive control substance(s):
yes
Remarks:
The sensitivity of the guinea-pig strain used is checked periodically at the testing facilities with known sensitisers hexyl cinnamic aldehyde (HCA), Benzocaine and mercaptobenzothiazole (MBT).
Positive control results:
A table with the results of the positive control tests of the last five years is part of the study report. The tests confirm the sensitivity of the strain and the procedure.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
7.5%
No. with + reactions:
25
Total no. in group:
25
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 7.5%. No with. + reactions: 25.0. Total no. in groups: 25.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
4%
No. with + reactions:
21
Total no. in group:
25
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 4%. No with. + reactions: 21.0. Total no. in groups: 25.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
7.5%
No. with + reactions:
25
Total no. in group:
25
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 7.5%. No with. + reactions: 25.0. Total no. in groups: 25.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
4%
No. with + reactions:
21
Total no. in group:
25
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 4%. No with. + reactions: 21.0. Total no. in groups: 25.0. Clinical observations: none.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
7.5%
No. with + reactions:
25
Total no. in group:
25
Clinical observations:
None
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 7.5% . No with. + reactions: 25.0. Total no. in groups: 25.0. Clinical observations: None.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
4%
No. with + reactions:
21
Total no. in group:
25
Clinical observations:
none
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 4%. No with. + reactions: 21.0. Total no. in groups: 25.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
4 and 7.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 4 and 7.5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
4 and 7.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 4 and 7.5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
4 and 7.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 4 and 7.5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Intradermal injections: Necrosis was recorded at sites receiving Freund’s Complete Adjuvant in test and control animals.

Slight irritation was seen in test animals at test item treated sites (0.5% w/w in water) for irrigation and no irritation was observed in control animals receiving water for irrigation.

Topical application: Slight erythema was observed in test animals following topical application with 10% w/w in distilled water. Similar signs of irritation were seen in the controls.

No dermal reactions were seen in all ten control group animals (induction with Freund`s adjuvant) challenged with eiter 7.5% or 4% of the test item.

Skin reactions were seen in all 25 test group animals.

Table 1: Challenge with 7.5% (L = Localised dermal reaction; * = Dryness and sloughin of the epidermis)

Animal no findings 24h 48h 72h
1 Erythema 2 2* 2*
edema 2 2 2
2 Erythema 2 2 2*
edema 2 2 2
3 Erythema 1L 1 1
edema 0 0 0
4 Erythema 2L 2L 2L
edema 0 0 0
5 Erythema 2L 2L 2L
edema 0 0 0
6 Erythema 2 2 2
edema 1 1 0
7 Erythema 2 2 2
edema 0 1 0
8 Erythema 2 2* 2*
edema 1 1 1
9 Erythema 2 2 2
edema 1 1 1
10 Erythema 2 2 2
edema 1 1 1
11 Erythema 2 2* 2*
edema 1 1 1
12 Erythema 1 2L 2L
edema 0 0 0
13 Erythema 2L 2L 2L
edema 0 0 0
14 Erythema 2 2* 2*
edema 1 1 1
15 Erythema 2L 2L 2
edema 0 0 0
16 Erythema 2 2* 2*
edema 1 1 1
17 Erythema 2 2 2*
edema 1 1 1
18 Erythema 2 2 2
edema 1 1 1
19 Erythema 1 1 1
edema 1 1 1
20 Erythema 2 2 2
edema 1 1 0
21 Erythema 2 2 2
edema 1 2 1
22 Erythema 1 1 1
edema 0 0 0
23 Erythema 2 2* 2*
edema 1 1 1
24 Erythema 2 2 2
edema 1 1 1
25 Erythema 2 2 2
edema 1 1 1

Table 2: Challenge with 4% (L = Localised dermal reaction; * = Dryness and sloughin of the epidermis)

Animal no findings 24h 48h 72h
1 Erythema 2 2* 2*
edema 1 1 1
2 Erythema 1 1 1
edema 0 0 0
3 Erythema 0 0 0
edema 0 0 0
4 Erythema 0 0 0
edema 0 0 0
5 Erythema 1L 1L 1L
edema 0 0 0
6 Erythema 1L 1L 1L
edema 0 0 0
7 Erythema 1 1 1
edema 0 0 0
8 Erythema 1L 1L* 1*
edema 0 0 0
9 Erythema 0 0 0
edema 0 0 0
10 Erythema 1L 1L 1L
edema 0 0 0
11 Erythema 1 1 1
edema 0 0 0
12 Erythema 1L 1 1
edema 0 0 0
13 Erythema 1L 1L 1L
edema 0 0 0
14 Erythema 1L 1L 1L
edema 0 0 0
15 Erythema 1L 1L 1
edema 0 0 0
16 Erythema 1 1 1
edema 0 0 0
17 Erythema 1 1 1
edema 0 0 0
18 Erythema 1 1 1
edema 1 1 0
19 Erythema 1L 1L 1L
edema 0 0 0
20 Erythema 1 1 1
edema 0 0 0
21 Erythema 1L 1 1L
edema 0 0 0
22 Erythema 0 0 0
edema 0 0 0
23 Erythema 1L 1 1
edema 0 0 0
24 Erythema 1 1 1
edema 0 0 0
25 Erythema 1 2 2
edema 0 0 0
Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

GHS Cat 1A (>60% guinea pigs sensitized with an intradermal induction concentration of 0.5% which is between 0.1- 1% )