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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Data was available before REACh was in place.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): pyrazolon
- Physical state: crystalline powder, light-beige
- Analytical purity: 100.5 g/100 g (H-NMR and IR spectroscopy)
- Storage condition of test material: room temperature, exclusion of light

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, Wiga, Kisslegg, Federal Republic of Germany
- Age at study initiation: young adults
- Weight at study initiation: 344 - 400 g
- Housing: 5/cage
- Diet (e.g. ad libitum): ad libitum (Kliba labor diet 341, assayed for chemical and microbiological contaminants)
- Water (e.g. ad libitum): ad libitum (tap water; about 2 g of ascorbic acid per 10l water was added to the drinking water twice a week, water is regularly assayed for chemical contaminants)
- Acclimation period: 7 days before the beginning of the study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C (deviations that would have had an adverse effect on the test results did not occur)
- Humidity (%): 30 - 70% (deviations that would have had an adverse effect on the test results did not occur)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
CMC (carboxymethyl cellulose)
Concentration / amount:
Vehicle: 1% CBC (Tylose CB 30000) in aqua bidest.

intradermal induction: 5% test substance preparation
percutaneous induction: 25% test substance preparation
1st and 2nd challenge: 10% test substance preparation
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
CMC (carboxymethyl cellulose)
Concentration / amount:
Vehicle: 1% CBC (Tylose CB 30000) in aqua bidest.

intradermal induction: 5% test substance preparation
percutaneous induction: 25% test substance preparation
1st and 2nd challenge: 10% test substance preparation
No. of animals per dose:
Control group 1: 5
Control group 2: 5 (second control group has been intended for a potential 2nd challenge in case of borderline results at the first challenge)
Test group: 10
Details on study design:
RANGE FINDING TESTS:
The concentrations of the test substance suitable for use in the main experiment were determined in the pretest.
The pretest was performed with 4 female animals for percutaneous and with 2 female animals for intradermal induction.

MAIN STUDY
A1. INDUCTION EXPOSURE - intradermal induction
- No. of exposures: 1
- Test groups:
A) front row : 2 injections each of 0 .1 ml Freund's adjuvant without test substance emulsified with 0 .9% aqueous NaCl-solution in a ratio of 1 : 1;
B) middle row : 2 injections each of 0 .1 ml of the test substance formulation;
C) back row : 2 injections each of 0 .1 ml Freund's adjuvant / 0 .9% aqueous NaCl-solution (1 : 1) with test substance
- Control group:
The animals were given the same injections (A, B, C) but without test substance, only with the formulating agent .
- Site: shoulder
- Frequency of applications: 1
- Concentrations: Test groups - 5% test substance preparations
- Readings: 24 h after the beginning of application, (scoring according to OECD 406)


A2. INDUCTION EXPOSURE - precutaneous induction
One week after intradermal induction
- No. of exposures: 1
- Test groups: 2 x 4 cm filter paper strips containing the test substance formulation were applied under an occlusive dressing
- Control group: 2 x 4 cm filter paper strips containing the solvent were applied under an occlusive dressing
- Site: shoulder, same area as in the case of the previous intradermal application
- Exposure time: 48 hours
- Frequency of applications: 1
- Concentrations: Test groups - 25% test substance preparations
- Readings: 48 hour after the beginning of application, (scoring according to OECD 406)

B. CHALLENGE EXPOSURE (1st and 2nd challenge)
- No. of exposures: 1
- Day(s) of challenge: 1st challenge - 14 days after the percutaneous induction; 2nd challenge - 21 days after the percutaneous induction (1 week after 1st challenge)
- Exposure period: 24 hours
- Test groups: 2 x 2 cm filter paper strips containing the test substance formulation were applied under an occlusive dressing
- Control group: 2 x 2 cm filter paper strips containing the test substance formulation were applied under an occlusive dressing
- Site: intact flank
- Concentrations: 10% test substance preparations
- Evaluation (hr after challenge): 24 and 48 h after the removal of the patch

1st challenge :
- The test group and control group 1 were treated with the test substance formulation . Additionally 1% solution of Tylose CB 30 .000 in aqua bidest .
was applied as a vehicle control to all animals . Control group 2 only received 1 % solution of Tylose CB 30 .000 in aqua bidest .
2nd challenge :
- The test group and control groups 1 and 2 were treated with the test substance formulation . Analogous to the lst challenge 1 % solution of
Tylose CB 30 .000 in aqua bidest . was applied as a vehicle control to all animals .

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: 1st reading, 1st challenge
Hours after challenge:
24
Group:
other: negative control 1
Dose level:
10%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
other: Reading:
Reading:
other: 2nd reading, 1st challenge
Hours after challenge:
48
Group:
other: negative control 1
Dose level:
10%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
other: Reading:
Reading:
other: 1st reading, 1st challenge
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: 2nd reading, 2nd challenge
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: 1st reading, 2nd challenge
Hours after challenge:
24
Group:
other: negative control 2
Dose level:
10%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
other: Reading:
Reading:
other: 2nd reading, 2nd challenge
Hours after challenge:
48
Group:
other: negative control 2
Dose level:
10%
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
other: Reading:
Reading:
other: 1st reading, 2nd challenge
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: 2nd reading, 2nd challenge
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading:

Any other information on results incl. tables

Induction:

After the intradermal induction well-defined erythema and slight edema were observed at the injection sites of all control group animals and all test group animals at which only Freund's adjuvant/0 .9% aqueous NaCl-solution (1 : 1) was applied . Injections of 5% test substance preparations in Tylose CB 30 .000, 1% in aqua bidest . or in Freund's adjuvant/0 .9% aqueous NaCl-solution (1 : 1) caused well-defined erythema and slight edema in all test group animals . The injection sites of all control group animals at which Tylose CB 30 .000, 1% in aqua bidest . was applied, did not show any skin reactions .

After the percutaneous induction with a 25% test substance preparation incrustation, partially open (caused by the intradermal induction), could be observed in addition to well-defined erythema and slight edema in all test group animals . The animals of control groups 1 and 2, which were applied with Tylose CB 30 .000, 1% in aqua bidest ., exhibited the same skin reactions as the animals of the test group .

1st Challenge:

After the lst challenge 1 out of 10 test group animals showed very slight erythema 24 hours after removal of the patches . 2 test group animals exhibited well-defined erythema . 1 out of these animals additionally showed scaling and very slight edema . Very slight erythema were observed in 3 out of 10 test group animals 48 hours after removal of the patches . 2 out of these animals additionally showed scaling .

The animals of control group 1 did not show any skin reactions . Tylose CB 30 .000, 1% in aqua bidest ., which was applied as a vehicle control to all animals, did not cause any skin reactions .

2nd Challenge:

After the 2nd challenge with a 10% test substance preparation no skin reactions were observed in all animals of the test group and control groups 1 and 2 24 and 48 hours after removal of the patches .

Tylose CB 30 .000, 1% in aqua bidest, which was applied as a vehicle control to all animals, did not cause any skin reactions .

Table 1: Skin findings after the challenge

1st challenge

2nd challenge

Test substance

(10% in vehicle)

Vehicle

Test substance

(10% in vehicle)

Vehicle

Control

group 1

0/4*

0/4*

0/4*

0/4*

Control

group 2

No application of test substance

0/4*

0/4*

0/4*

Test group

3/10*

0/10*

0/10*

0/10*

* number of positive reactions/number of animals tested (reading at 24h and/or at 48 hours after removal of the patch)

Body weights:

The expected body weight gain was generally observed in the course of the study .

Mortality:

1 animal of control group 1 and 1 animal of control group 2 died 10 days after the beginning of the study . Macroscopic examination revealed that the animals suffered from pneumonia . The cause of death was not related to the test substance treatment .

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met