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Diss Factsheets

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: MAGNUSSON B. - KLIGMAN A.M. The identification of contact allergens by animal assay "The guinea pig maximization test". J. Invest. Derm. 1969,52,3,268-276
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
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
Reference substance name:
Reaction products of 6-amino-4-hydroxynaphthalene-2-sulphonic acid and 7-amino-4-hydroxynaphthalene-2-sulphonic acid, condensed with Formaldehyde and Sodium hydrogensulfite, coupled with diazotised Sodium 2-amino-4,6-dinitrophenoxide, metalized with Sodium Dichromate, sodium salts
EC Number:
947-452-3
Molecular formula:
not applicable
IUPAC Name:
Reaction products of 6-amino-4-hydroxynaphthalene-2-sulphonic acid and 7-amino-4-hydroxynaphthalene-2-sulphonic acid, condensed with Formaldehyde and Sodium hydrogensulfite, coupled with diazotised Sodium 2-amino-4,6-dinitrophenoxide, metalized with Sodium Dichromate, sodium salts
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: C.E.R.J. (53940 Saint Berthevin, France)
- Age at study initiation:
- Weight at study initiation:
Control group: 405 g for the males and 367 g for the females
Test group: 401 g for the males and 367 g for the females
- Housing: The animals were housed individually in polypropylene cages (O,43 x 0,23 x 0,17 m). Each cage was equipped with a food container and a water bottle.
The floor of the cages contained wooden saw dust (Société Parisienne des Sciures, 93500 Pantin, France).
Wooden saw dust chemical analysis was carried out periodically by the Laboratoire Municipal de Rouen (76000 Rouen, France).
- Diet: the animals were fed ad libitum during the study, with a certified pelleted diet Rabbit diet S.Q.C. (Special Diets Services Ltd., Nitham, Essex, England).
- Water: the animals had free access to bottles containing tap water filtered with Millipore filters (0,22 micron) and supplemented with vitamin C. Bacteriological and chemical water analysis and detection for major contaminants were performed regularly by the Laboratoire Municipal de Rouen (76000 Rouen, France).
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3 °C
- Humidity: 50 ± 20 %
- Air changes: The animals were maintained in an air-conditioned room. The incoming, non-recycled air, was filtered with an absolute filter.
- Photoperiod: The light/dark cycle was 12 hours per day

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Concentration / amount:
2.5 %
Day(s)/duration:
day 0
Route:
intradermal
Vehicle:
other: 50/50 (v/v) mixture of Freund's complete adjuvant at 50 %; (v/v) in saline (NaCl 9 %) and vehicle (distilled water).
Concentration / amount:
2.5 %
Day(s)/duration:
day 0
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 g
Day(s)/duration:
day 8 for 48 hours
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 g
Day(s)/duration:
day 25 for 24 hours
No. of animals per dose:
10 per sex per dose
Details on study design:
RANGE FINDING TESTS: yes

MAIN STUDY
-INDUCTION EXPOSURE: Intradermal route
- No. of exposures: 3 x 2 intradermal injections 0.1 ml each
- Exposure period: day 0
- Test groups: 20 animals (10 ♂ - 10 ♀)
- Control group: 10 animals (5 ♂ - 5 ♀)
- Site: on the scapula on a surface area of 4 x 2 cm (one injection of each solution per flank).
- Frequency of applications:
- Concentrations:
Test group:2.5 %
Control group: vehicle only
-Vehicle: A 50/50 (v/v) mixture of Freund's complete adjuvant diluted at 50 % in saline (NaCl 9 0/00)

-INDUCTION EXPOSURE: Epicutaneous
-No. of exposures: one
-Day(s) of challenge: day 8
- Test groups: 20 animals (10 ♂ - 10 ♀)
- Control group: 10 animals (5 ♂ - 5 ♀)
-Site: scapular region
-Concentrations:
Test group: 0.5 g
Control group: 0.5 ml of the vehicle
Vehicle:
Test group: no vehicle
Control group: 0.5 ml of distilled water
-Evaluation (hr after challenge): 48 h
-Other: On Day 7; 0.5 ml of sodium lauryl sulfate 10 % in codex vaseline were applied on the scapular region to provoke a local irritation.

B. CHALLENGE EXPOSURE
- No. of exposures:
- Day(s) of challenge: 25
- Exposure period:
- Test groups:
- Control group:
- Site: flanks
- Concentrations:
Test group: 0.5 g undiluted test sample
Control group: 0.5 ml of the vehicle (distilled water)
- Evaluation (hr after challenge): 24 h
-Other: occlusive patch tests were applied on 4 cm². The patches were kept in place by an hypo-allergenic tape. The right flank was used as negative control.
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:
2.5 % in distilled water (intradermal induction)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no change in behaviour nor clinical signs due to treatment were observed during the study.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
2.5 % in distilled water (intradermal induction)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no change in behaviour nor clinical signs due to treatment were observed during the study.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no change in behaviour nor clinical signs due to treatment were observed during the study
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no change in behaviour nor clinical signs due to treatment were observed during the study
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

Mortality

A female of the control group I was found dead on Day 8.

Macroscopic and microscopic observation

Individual macroscopic and microscopic observations are given thereafter.

1. Macroscopic observations

No oedematous reactions were observed on the challenge test site 24 and 48 hours after removal of the patch.

The redness of the skin was not scored due to the staining of the skin by the test sample.

2. Microscopic observation:

Skin reactions to the application of the test article or solvent were noted on animals from both groups I and II.

In group I :

3 animals (333, 334, 335) showed minimal skin reactions on their left flank.

In group II :

2 animals (343, 352) showed minimal skin reactions on their right flank.

6 animals (340, 341, 344, 353, 360, 361) showed minimal skin reactions on both flanks.

6 animals (338, 339, 345, 354, 355, 358) showed minimal skin reactions only on their left flank.

The intensity of reactions was minimal in every case and the primary lesions were those skin irritation : minimal hyperkeratosis, minimal hyperacanthosis, foci of acantholysis, foci of spongiosis, foci of oedema and inflammatory cells in the upper dermis.

Due to the test-article formula or to the staining process, there was no evidence of colouring of the stratum corneum. Only black material deposits could be seen on all treated flanks.

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP classification (EC 1272/2008)
Conclusions:
The test sample did not provoke any cutaneous sensitization in the guinea pig, under our experimental conditions.
Executive summary:

The sensitizing potential of substance was tested after intradermal injections and cutaneous applications in ten male and ten female Albino Guinea Pigs, to the method established by B. Magnusson and A. Kligman *.

The intradermal induction was performed at the concentration of 2.5 % in distilled water. The epicutaneous induction and the challenge patch test were performed with the undiluted test sample. The behaviour of the animals was not influenced by the treatment. No local reaction could be observed 24 h and 48 h after removal of the challenge patch test due to the staining of the skin. Cutaneous samples of both flanks were taken and fixed in formalin 10 %. After histological examination, there was no evidence of a sensitization due to the test sample.

In conclusion, under our experimental conditions, the test sample did not provoke any cutaneous sensitization in the guinea pig.

 

* MAGNUSSON B. - KLIGMAN A.M. The identification of contact allergens by animal assay "The guinea pig maximization test". J. Invest.Derm. 1969,52,3,268-276