2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.04.2003 - 29.07.2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted JuIy 17, 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

JuIy 30, 1996

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This study was performed prior to the EU REACH legislation and the LLNA was not yet the primary method to be used.

Test material

Test material information

Constituent 1

Reference substance name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

EC Number:

278-388-8

EC Name:

2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Cas Number:

76199-85-4

Molecular formula:

C16H11N5O4

IUPAC Name:

2-cyano-N-methyl-2-[3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)-2,3-dihydro-1H-isoindol-1-ylidene]acetamide

Details on test material:

- Test substance No.: 03/0047-1
- Physical state: Powder / yellow
- Analytical purity: 94.7 %
- Stability under test conditions: The analytical characterization was run almost in parallel to the administration phase of the study. Therefore the results of this analysis represent the composition of the test substance and no reanalysis is necessary.
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Hsd Poc: DH (SPF)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: About 5 weeks
- Weight at study initiation: 260 g - 337 g
- Housing: 5 animals per cage, stainless steel wire mesh cages with plastic-coated grating, minimum floor area: 2000 cm²,
- Diet: Kliba Labordiaet (Kaninchen/ Meerschweinchen-Haltungsdiaet) Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 6 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

CMC (carboxymethyl cellulose)

Induction 2

Concentration / amount:

5% test substance in 1% CMC for intradermal induction

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

CMC (carboxymethyl cellulose)

Challenge 2

Concentration / amount:

25% test substance in 1% CMC for epicutaneous induction and challenge

No. of animals per dose:

test group: 10, control group: 5

Details on study design:

PREPARATION OF THE TEST SUBSTANCE FORMULATIONS
The test substance preparations were produced on a weight by weight (w/w) basis shortly before the application by stirring with a high speed homogenizer (Ultra-Turrax) and a magnetic stirrer.
The homogeneity of the test substance preparation during application was provided by stirring with a magnetic stirrer or a spatula.
Form of test substance preparations:
The test substance was applied as a suspension for all applications.
The maximum suitable concentration tor application was 25%, due to its highly viscous, pasty consistence.

SELECTION OF THE VEHICLE
1 % CMC (cleaned sodium carboxymethylcellulose) in doubly distilled water was used as the vehicle because good homogeneity of the preparation was achieved.

RANGE FINDING TESTS:
The concentrations for the test substance suitable for use in the main experiment were determined in the pretest.
One 24-hour epicutaneous occlusive application was performed. 10% and 25% test substance preparations in 1 % CMC-solution in doubly distilled water were tested. No skin irritation could be noticed (findings 24 and 48 hours after removal of the patch).
Applicability: it was possible to inject a 5% test substance preparation in 1 % CMC-solution in doubly distilled water or 5% in Freund's adjuvant / 0.9% aqueous NaCI-solution (1 : 1) with a syringe. The concentration was weIl-tolerated Iocally and systemically.
The following concentrations for induction and the challenge were selected on the basis
of the pretest:
- Intradermal induction: Test substance 5% in 1 % CMC-solution in doubly distilled water or 5% in Freund's adjuvant / 0.9% aqueous NaCI-solution (1:1)
- Epicutaneous induction: Test substance 25% in 1 % CMC-solution in doubly distilled water
- Challenge: Test substance 25% in 1 % CMC-solution in doubly distilled water

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2, intradermal induction (6 intradermal injections in groups of two per animal were given): day 0; Epicutaneous induction: day 6
- Intradermal induction:
- Test groups: Test substance 5% in 1 % CMC-solution in doubly distilled water or 5% in Freund's adjuvant / 0.9% aqueous NaCI solution (1:1); Epicutaneous induction: Test substance 25% in 1 % CMC-solution in doubly distilled water
A) front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCI-solution in a ratio of 1 : 1
B) middle row: 2 injections each of 0.1 ml of a test substance formulation in an appropriate vehicle at the selected concentration
C) back row: 2 injections each of 0.1 ml Freund's adjuvant /0.9% aqueous NaCI-solution (1 : 1) with test substance at the selected concentration.
- Control group:
A) front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCI-solution in a ratio of 1 : 1
B) middle row: 2 injections each of 0.1 ml of the undiluted vehicle
C) back row: 2 injections each of 0.1 ml of a 50% formulation of the vehicle without test substance emulsified with Freund's adjuvant /0.9% aqueous NaCl-solution (1:1)
- Site: neck region
- Readings: 24 h after the beginning of application

EPICUTANEOUS INDUCTION:
- amount applied: 2 x 4 cm gauze patches (6 Iayers surgical gauze Ph. Eur. from Lohmann GmbH & Co.KG) containing the test substance formulation were applied to the skin of the neck region under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 6 cm from Russka) and Fixomull®Stretch (adhesive fleece) from Beiersdorf AG.
1 g of the test substance formulation was applied to each animal.
- The control animals were not treated since the 1 % CMC-solution in doubly distilled water used as formulating agent was not expected to influence the result of the study.
- Duration of exposure: 48 hours
- Site of application: neck region, same area as in the case of the previous intradermal application
- Readings: 48 h after the beginning of application

B. CHALLENGE EXPOSURE
The challenge was performed 14 days after the epicutaneous induction.
- No. of exposures: 1
- Amount applied: 2 x 2 cm gauze patches (6 Iayers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test substance formulation were applied to the skin of the flank under an occlusive dressing. The dressing c onsisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (5 x 5 cm from Pfaelzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
0.5 g of the test substance formulation was applied to each animal.
Ihe test group and the control group were treated with the test substance formulation.
- Duration of exposure: 24 hours
- Site of application: intact flank
- Removal of the test substance: with water / REDURAN® (special cleanser for dyes, Stockhausen, Krefeld)
- Evaluation (hr after challenge): 24 and 48 h after the removal of the patch

Challenge controls:

5 female animales, dose for challenge application: 0.5 g

Positive control substance(s):

yes

Remarks:

5% (intradermal inductuction and challenge), 10% (epicutaneous induct.) Alpha-Hexylcinnamaldehyde techn. 85% in Lutrol E400 (epicutanous induct. and challenge) /in paraffin oil resp. or Freund's adjuvant/0.9% aqu. NaCl-solution (1:1) (intradermal induct.)

Results and discussion

Positive control results:

The positive control shows that Alpha-Hexylcinnamaldehyde techn. 85% has a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen and is therefore able to detect sensitizing compounds.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% in 1% CMC-solution in doubly distilled water

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

Grade 1 (discrete or patchy erythema) according to the grading scale of Magnusson and Kligman (max. score 3)

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25% in 1% CMC-solution in doubly distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no skin reactions

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25% in 1% CMC-solution in doubly distilled water

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no skin reactions

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25% in 1% CMC-solution in doubly distilled water

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no skin reaction

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study it was concluded that the test substance does not have a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test substance (purity: > 94.7 weight-%) was assessed based on the method of Magnusson and Kligman in a GLP-conform study conducted according to OECD guideline 406. Based on the results of a pretest and given that the test substance was highly viscous and pasty the maximal applicable concentration was 25%. The intradermal induction was performed with a 5 % test substance preparation in 1 % aqueous CMC-solution or 5 % test substance preparation in Freund's adjuvant/ 0.9 % aqueous NaCl-solution (1 : 1). The epicutaneous induction as well as the challenge were performed with a 25 % test substance preparation in 1 % aqueous CMC-solution.

The study was conducted with one control group (5 female animals) and one test group (10 female animals). The intradermal induction was performed on day 0, the epicutaneous induction on day 6 and the challenge was carried out 14 days after the epicutaneous induction.

The intradermal induction caused moderate and confluent to intense erythema and swelling at the injection sites of the test substance preparation in all test group animals. After the epicutaneous induction incrustation, partially open (caused by the intradermal induction) could be observed in addition to moderate and confluent erythema and swelling in all test group animals. The challenge caused discrete or patchy erythema in two test group animals, 24 hours after removal of the patch, but was reversible within 48 hours.

Based on the results of this study it was concluded that the test substance does not have a sensitizing effect on the skin of the guinea pig in the Maximization Test under the test conditions chosen.