Reaction product of: 2,4-diamino-6-[2-(2-methyl-1H-imidazol-1-yl)ethyl]-1,3,5-triazine and cyanuric acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Jun to 27 Jun 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Adopted 24 Apr 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

EEC Methods for the determination of toxicity. Annex to Commission Directive 2004/73/EC (Official Journal No. L152, 30.4.2004), Part B

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, London, England

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: harlan UK Ltd., Bicester, Oxon, England
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: not specific pathogen-free (SPF) animals
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 16.1 to 22.6 g
- Housing: housed individually in polycarbonate cages with woodflake bedding, provided Nestlets for environmental enrichment
- Diet: Special Diet Services RMI (E) SQC, ad libitum
- Water: municipal drinking water, ad libitum
- Acclimation period: at least six days
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 40 to 70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: 12 Jun 2006 To: 27 Jun 2006

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

10, 20, 50% w/v

No. of animals per dose:

4

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Lymph node 3H-methyl Thymidine (3HTdR) incorporation determined by Β-scintillation counting.
- Criteria used to consider a positive response: The test substance is condsidered as a sensitizer if at least one concentration of the test substance results in a three-fold greater increase in 3HTdR incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION: Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the entire dorsal surface of each ear for three consecutive days (Days 1-3) using a micropipette for evenly-spread application. A further two groups of four mice received the vehicle alone or the positive control substance (HCA). All animals were observed daily for signs of toxicity and local ear irritations were assessed. The maxiumum practical concentration for pinna dosing was 50% (w/v). Five days following the first topical application of the test substance (Day 6), all mice were injected via tail vein with 250 µL of PBS containing 3HTdR giving a nominal dose of 20 µCi to each mouse. Five hours later, the draining auricular lymph node of each ear was excised into PBS, lymph nodes being pooled per treatment group. A single cell suspension of lymph nodes (LNC) was prepared by gentle mechanical disaggregation, washed, pelleted (by centrifugation), and resuspended three times in PBS. The final resuspension was in trichloroacetic acid (TCA: 5%). Macromolecules were precipitated with 5% TCA at 4 °C overnight. The precipitate was pelleted by centrifugation, resuspended in 5% TCA, added to Ultima Gold scintillation cocktail and measured by B-scintillation counting.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Body weight means and test/control ratios were calculated.

Results and discussion

Positive control results:

The test/control ratio for the HCA-treated group was 9.8. A three-fold greater increase compared to control values indicates a sensitizer. This test/control ratio value showed the effectiveness of the positive control treatment.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

other: Test/control ratio

Value:

0.8

Variability:

pooled results per group

Test group / Remarks:

10% (w/v)

Results 2

Key result

Parameter:

other: Test/control ratio

Value:

1.2

Variability:

pooled results per group

Test group / Remarks:

20% (w/v)

Results 3

Key result

Parameter:

other: Test/control ratio

Value:

1.4

Variability:

pooled results per group

Test group / Remarks:

50% (w/v)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Disintegrations per minute (DPM):

Group Concentration % (w/v) DPM Number of lymph nodes DPM/node Test/control (+) = positive
ratio (-) = negative

1 DMSO 2510.70 8 313.84 NA NA
2 10 2047.50 8 255.94 0.8 (-)
3 20 2910.00 8 363.75 1.2 (-)
4 50 3630.90 8 453.86 1.4 (-)
5 HCA 24646.90 8 3080.86 9.8 (+)

CLINICAL OBSERVATIONS: There were no deaths or signs of ill health or toxicity observed during this study. Reddening of the skin around the cranial area was noted after dosing in three animals in Group 5 (HCA) on Day 3 (final application), this had resolved by Day 4.

BODY WEIGHTS: One female (Group 2) lost body weight during the study. All remaining animals gained weight during the study.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008