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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:
June 23, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
GLP compliance:
yes
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
Chemical structure
Reference substance name:
Reaction products of diazotised 3-amino-2-hydroxy-5-nitrobenzenesulphonic acid, coupled with 1,3-diaminobenzene and diazotised sodium 4-aminobenzenesulfonate, metallised with Basic Chromium (III) Sulphate
EC Number:
947-395-4
Molecular formula:
not applicable
IUPAC Name:
Reaction products of diazotised 3-amino-2-hydroxy-5-nitrobenzenesulphonic acid, coupled with 1,3-diaminobenzene and diazotised sodium 4-aminobenzenesulfonate, metallised with Basic Chromium (III) Sulphate
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
-Source: David Hall Limited, Burton-on-Trent, Staffordshire, U.K
-Age at study initiation: 8-10 weeks
-Weight at study initiation: 305 - 420 g
-Housing: the animaìs were housed in groups of up to four in solid-floor polypropylene cages furnished with softwood shavings.
-Diet: free access to food (Guinea Pig FDI Diet, Special Diet Services Limited, Witham, Essex, U.K.)
-Water: free access to mains tap water
-Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
-Temperature: 20 - 24 °C
-Humidity: 60 - 70 %
-Air changes: 15 changes per hour
-Photoperiod: 12 hours cycle dark/light

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Concentration / amount:
5% (w/v)
Day(s)/duration:
day 0
Route:
intradermal
Vehicle:
other: Freund's Complete Adiuvant plus distilled water in the ratio 1:1
Concentration / amount:
5 % (w/v)
Day(s)/duration:
day 0
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
75% (w/w)
Day(s)/duration:
day 7 for 48 hours
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
25% (w/w)
Day(s)/duration:
day 21 for 24 hours
No. of animals per dose:
PRETEST: 2 per 4 concentrations
TEST GROUP: 20
CONTROL GROUP: 10
Details on study design:
RANGE FINDING TESTS
INDUCTION EXPOSURE: INTRADERMAL
Suspensions of the test material in distilled water were tested to determine the highest practicaì level (up to 25 %) that could be intradermally injected and well tolerated both locally and systemically.
INDUCTION EXPOSURE: EPICUTANEOUS
Suspensions of the test material in distilled water were tested to determine the hìghest practical level which did not, produce excessive inflammation on the flank of animals injected with Freund's Complete Adjuvant (Difco Laboratories, Michigan, U.S.A.) at least seven days previously.
CHALLENGE EXPOSURE: EPICUTANEOUS
Suspensions of the test material in distilled water were tested to determine the highest practical level which did not produce inflammation or irritation on the flank of animals iniected with Freund's Complete Adjuvant at least seven days previously.

MAIN STUDY
INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: 3 x 2 intradermal injections 0.1 ml each
- Exposure period: day 0
- Test groups: 20 females animals
- Control group: 10 females animals
- Site: 40 mm x 60 mm on the shoulder region
- Frequency of applications: single injection
- Concentrations:
Test group
1° site) Freund's Complete Adiuvant plus distjlled water in the ratio 1:1
2° site) 5.0% (w/v) suspension of test material in distilled water
3° site) 5.0% (w/v) suspension of test material in a 1:1preparation of Freund's Complete Adiuvant plus distilled water.
Control group
1° site) Freund's Complete Adiuvant plus distjlled water in the ratio 1:1
2° site) Distilled water
3° site) Freund's Complete Adiuvant plus distilled water.
INDUCTION EXPOSURE: EPICUTANEOUS
-No. of exposures: one
-Day(s) of challenge: day 7
-Test groups: 20 females animals
-Control group: 10 females animals
-Site: 40 mm x 60 mm on the shoulder region
-Concentrations:
Test group: 0.2-0.3 ml at concentration of 75% (w/w)
Control group: 0.5 ml of the vehicle
-Evaluation (hr after challenge): 48 h
-Other: on tested site was covered with a filter paper (WHATMAN No. 4: approximate size 40 mm x 20 mm) which was held in place by a strìp of surgical adhesive tape (BLENDERM: approximate size 60 mm x 25 mm) and covered with an overlapping length of aluminium foil. The patch and foil were further secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal

CHALLENGE EXPOSURE
-No. of exposures: one
-Day(s) of challenge: 24
-Exposure period: 24
-Test groups: 20 females animals
-Control group: 10 females animals
-Site: area, approxìmately 50 - 70 x 50 mm on flank
-Concentrations:
Test group
0.1 - 0.2 ml of the test material formulation (25%w/w in distilled water) was applied to the shorn right flank
Control group
Vehicle only
-Other: on tested site was covered with a filter paper (WHATMAN No. 4: approximate size 20 mm x 20 mm) which was held in place by a strìp of surgical adhesive tape (BLENDERM: approximate size 40 x 50 mm). The patches were occluded with an overlappìng length of aluminium foil and secured by a strip of elastic adhesive bandage (ELASTOPLAST: approxìmate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.
Positive control substance(s):
yes
Remarks:
2,4-DINITROCHLOROBENZENE (DNCB) in a validate study (April 1989)

Results and discussion

Positive control results:
The reference substance produced a 79 % (15/19) sensitisation rate. This was considered to be a satisfactory sensitisation response for this material under the conditions of the test.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5 % in distilled water (intradermal induction)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No adverse reactions were noted at the test material and vehicle control sites of the test or controì animals at the 24 and 48-hour observations.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5 % in distilled water (intradermal induction)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No adverse reactions were noted at the test material and vehicle control sites of the test or controì animals at the 24 and 48-hour observations.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No adverse reactions were noted at the test material and vehicle control sites of the test or controì animals at the 24 and 48-hour observations.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No adverse reactions were noted at the test material and vehicle control sites of the test or controì animals at the 24 and 48-hour observations.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1 % (w/v) (intradermal induction)
No. with + reactions:
15
Total no. in group:
19
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1 % (w/v) (intradermal induction)
No. with + reactions:
9
Total no. in group:
19

Any other information on results incl. tables

PRETEST

-Toxicological signs

Signs of systemic toxicity (lethargy, pallor,  hunched  posture and emaciation) were noted in the animal intradermally injected with the test material at a concentration  of  25% (w/v) in distilled water. This animal was killed for humane reasons 48 hours after injection.

Animals injected wìth the test material at a concentration  of l%, 5% or l0% showed no signs of systemic toxicity.

Post mortem examination

Necropsy revealed severe oedema extending  over the nuchal, thoracic, abdominal and cervical regions and around the front limbs, accompanied by brown/black  discoloration of the subcutaneous  tissue.

-Main Study

a) Induction

Brown-coloured staining caused by the test material was noted over the whole treatment site following  patch removal after the topical induction period. This did not preclude accurate assessment of the dermal responses.

Scattered mild redness was commonly noted at the test material sites of the test group animals at the one hour observation. Scattered mild or moderate and diffuse redness persisted at the test material sites at the 24 -hour observatjon. No adverse skin reactions were elicited by the vehicle.

b) Challenge

Faint brown-coloured staining  was noted at the test material sites of three test group animals at challenge. This did not preclude accurate assessment of the skin responses.

No adverse reactions were noted at the test material and vehicle control sites of the test or controì animals at the 24 and 48-hour observations.

-Bodvweight

Bodyweight  gains of guinea pigs in the test group, between day 0 and day 24, were comparable to those observed  in the control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP regulation (EC) no. 1272/2008
Conclusions:
The test material was not found to be a sensitiser to guinea pig skin, under the test condition.
Executive summary:

In order to assess the skin sensitisation potential of the test material an in vivo test to guinea pigs was performed according to the OECD Guideline 406 (1981).

Based on the results of the sighting studies, the concentrations of test material for the induction and challenge phases were selected  as follows:

Intradermal Induction: 5% (w/v) in distilled water

Topical Induction: 75% (w/w) in distilled water

Topical Challenge: 25% (w/w) in distilled water

The test material produced a 0 % (0/20) sensitisation rate and was not considered as a sensitiser to guinea pig skin.