N-nitro-N-(3-methyl-3,6-dihydro-2H-1,3,5-oxadiazin-4-yl)amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-09-11 till 1996-01-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-060-1

EC Name:

-

Cas Number:

153719-38-1

Molecular formula:

Hill formula: C4H8N4O3 CAS formula: C4H8N4O3

IUPAC Name:

3-methyl-N-nitro-3,6-dihydro-2H-1,3,5-oxadiazin-4-amine

Details on test material:

- Substance type: mono-constituent organic substance
- Physical state: solid, fine, white powder
- Analytical purity: 96.7 %
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: P.503005
- Expiration date of the lot/batch: Date of reanalysis: December 1996
- Stability under test conditions: not reported
- Storage condition of test material: 0 - 5 °C

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Limited, Laboratory Animal Breeding, Pharma Division, 4332 Stein / Switzerland
- Age at study initiation: not reported
- Weight at study initiation: 323 to 406 g
- Housing: The animals were housed individually in Macrolon cages (Type 3)
- Diet (e.g. ad libitum): The animals received ad libitum standard guinea pig pellets - NAFAG 845, NAFAG, Gossau/SG, Switzerland
- Water (e.g. ad libitum): fresh water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light cycle day


IN-LIFE DATES: From: 1995-11-13 To: 1955-10-12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

peanut oil

Induction 2

Concentration / amount:

Concentration of test material and vehicle used at induction:
a) Injection into the skin: 1 % test substance in peanut oil
b) Application to the skin: 50 % test substance in vaseline

Concentration of test material and vehicle used for each challenge: 10 % test item in vaseline.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

peanut oil

Challenge 2

Concentration / amount:

Concentration of test material and vehicle used at induction:
a) Injection into the skin: 1 % test substance in peanut oil
b) Application to the skin: 50 % test substance in vaseline

Concentration of test material and vehicle used for each challenge: 10 % test item in vaseline.

No. of animals per dose:

Number of animals in test group: 10 male and 10 female
Number of animals in negative control group: 10

Details on study design:

RANGE FINDING TESTS:

A. Intradermal Induction:
The vehicle, peanut oil, was selected with respect to the solubility of the test article and lack of sensitising properties. The concentration for intradermal injections was selected on account of the solubility of the test article in standard vehicles, the local irritant potency and the systemic tolerability of the test article in a pretest.
The following test item concentrations were examined in pretest animals in duplicate: 1, 3 and 5% (w/v) in peanut oil.
Local reactions at the injection sites were examined 48 hours after duplicate intradermal injections.

B. Epidermal Applications (induction and challenge)
The concentration for the epidermal applications was selected on account of the primary irritation potential of the test article.
The following test item concentrations were examined in pretest animals in duplicate to determine the maximum subirritant concentration: 10, 30 and 50% (w/v) in vaseline.
At least 7 days before application of the test item, two pairs of consecutive intradermal injections of an adjuvant/saline mixture 1:1 (v/v; 0.1 ml per injection) were applied to the shaved neck of 2 guinea pigs. On each animal, 3 different test item concentrations were tested simultaneously on the left and right flank using Hilltop chambers. Each concentration was tested in duplicate, naive skin site served as control. Occlusive dressing was applied for 24 hours. Skin responses were scored 24 and 48 hours after removal of the dressing according to the Draize scale.


MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures:
Intradermal: three pairs of intradermal injections (0.1 ml per injection) were made consecutively on the left and right side of the shaved neck in test and control group animals.
Epidermal exposure: Test item was suspended in vaseline and applied on a filter paper patch to the neck of the animals (patch 2x4 cm; approx. 0.4 g per patch; occlusive dressing).
- Exposure period: intradermal injection: exposure period throughout the whole test; epidermal exposure: 48 h
- Test groups:
Intradermal injection: (1) adjuvant/physiological saline mixture 1:1 (v/v), (2) 1% test item in peanut oil, (3) 1% test item in the adjuvant/physiological saline mixture (w/v)
Epidermal induction: 50% test item in vaseline
- Control group:
Intradermal injection: (1) adjuvant/physiological saline mixture 1:1 (v/v), (2) adjuvant/physiological saline mixture 1:1 (v/v), (3) peanut oil
Epidermal induction: vaseline
- Site: left and right side of the neck
- Frequency of applications: once only
- Duration: total test duration removal of epidermal induction patch on day 10 (start epidermal induction on day 8)
- Concentrations: injection: 1% test item in peanut oil; epidermal: 50% test item in vaseline


B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day(s) of challenge: day 22
- Exposure period: 24 h
- Test groups: 10% test item in vaseline
- Control group: vaseline only
- Site: test item with vaseline on one flank, vaseline anly at the other flank
- Concentrations: 10% test item in vaseline
- Evaluation (hr after challenge): The challenge reactions were scored 24 and 48 hours after removing the dressings according to the Draize scale.


OTHER:

Challenge controls:

adjuvant/physiological saline mixture 1:1 (v/v)

Positive control substance(s):

yes

Remarks:

hexyl cinnamic aldehyde

Study design: in vivo (LLNA)

Statistics:

no statistics applied

Results and discussion

Positive control results:

The results of the latest positive control test are presented in Appendix 3 of the original study report.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10 %

No. with + reactions:

6

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 6.0. Total no. in groups: 20.0.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10 %

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

Very slight erythema reactions in 1 male and 1 female faded after the scoring at 24 hours and were recognised as transient irritation (false-positive reaction)

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: Very slight erythema reactions in 1 male and 1 female faded after the scoring at 24 hours and were recognised as transient irritation (false-positive reaction).

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10 %

No. with + reactions:

4

Total no. in group:

20

Clinical observations:

Scaling was recorded in 2 male animals after 48 hours.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 %. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: Scaling was recorded in 2 male animals after 48 hours..

Results 4

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

The sensitization rate was 30% including transient irritant reactions.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Based on the sensitization rate of 30% including transient irritant reactions, which is at the threshold of significance set in OECD-GHS as well as in Council Directive 67/548/EEC, test item CA 2343 A (Intermediate of CGA 293343) is a skin sensitizer and thus has to be labelled "May cause sensitization by skin contact".

Executive summary:

A guinea pig maximisation test was initiated to determine sensitising properties of test item CA 2343 A (Intermediate of CGA 293343) after challenge exposure by skin contact. The procedure of Magnusson and Kligman for adjuvant tests was followed.

Epidermal challenge of 10 male and 10 female guinea pigs (test groups) resulted in positive responses in 6 (3 males, 3 females) animals after 24 hours and in 4 (2 males, 2 females) animals after 48 hours. Very slight erythema reactions in 1 male and 1 female faded after the scoring at 24 hours and were recognised as transient irritation (false-positive reaction) based on clinical appearance and duration. Scaling was recorded in 2 male animals (No. 126 and 134) after 48 hours. No irritant skin reactions were recorded for control animals. The sensitization rate was 30% including transient irritant reactions. Body weights were not affected by the treatment.

Based on the sensitization rate of 30% including transient irritant reactions, which is at the threshold of significance set in OECD-GHS as well as in Council Directive 67/548/EEC, test item CA 2343 A (Intermediate of CGA 293343) is a skin sensitizer and thus has to be labelled "May cause sensitization by skin contact".