2,2`-[6-(phenylamino)-heteromonocycle-2,4-diyl]diphenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 24 April 2002

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2,2`-[6-(phenylamino)-1,3,5-triazine-2,4-diyl]diphenol

Cas Number:

1248-66-4

Molecular formula:

C21H16N4O2

IUPAC Name:

2,2`-[6-(phenylamino)-1,3,5-triazine-2,4-diyl]diphenol

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks (beginning of acclimatisation)
- Housing: single in makrolon Type I, with wire mesh top
- Diet: pelleted standard diet ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-76%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

6.25, 12.5 and 25% (w/v)

No. of animals per dose:

4 females (nulliparous and non-pregnant)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 25% suspension in dimethylformamide (highest technically applicable concentration)
- Irritation: treatment with 25% suspension did not induce irritation ;

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by a stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high dose concentrations) for ether local toxicity or immunological suppression;

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 6.25, 12.5 and 25 % (w/v) in dimethylformaide. The application volume, 25 µl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Five days after the first topical application, all mices were administered with 250µl of 81.1 µCi/ml 3HTdR (corresponds to 20.3 µCi 3HTdR per mouse) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions were prepared, washed with phosphate buffered saline, resuspended in trichloroacetic acid, incubated for 18 h at 4°C;
The precipitates were than resuspended in 5% trichloroacetic acid (1ml) and transferred to plastic scintillation vials with 10 ml of "Ultima Gold" scintillation liquid and thoroughly mixed.
the 3HTdR incorporation was measured on a .beta.-scintillation counter.

Positive control substance(s):

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

Statistics:

the mean values and standard deviations were calculated for body weights

Results and discussion

Positive control results:

vehilcle: acetone: olive oil (4+1);
test item concentration (w/v), DPM lymph node, S.I.
5% , 936.1, 2.43
10 %, 1569.0, 4.07
25%, 1883.2, 4.88

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

6.42

Test group / Remarks:

6.25% in vehicle

Results 2

Parameter:

SI

Value:

5.48

Test group / Remarks:

12.5% in vehicle

Results 3

Parameter:

SI

Value:

6.33

Test group / Remarks:

25% in vehicle

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per lymph node: control: 1036.7 6.25 %: 6656.7 12.5 %: 5677.1 25 %: 6557.8

Applicant's summary and conclusion

Interpretation of results:

other: ambigous

Conclusions:

The test item was found to be a sensitizer under the described conditions;
however as there was no dose response the test result is somewhat ambigous; moreover a structurally very closely related substance is not classified as a sensitizer based on a guinea pig sensitizing test. Due to these diverting results it was decided to perform a G.P.M.T. to clarify the result of the LLNA;

Executive summary:

In the study the test item suspended in dimethyl formamide was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 6.25, 12.5, and 25%. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In the present study Stimulation Indices (S.I.) of 6.42, 5.48, and 6.33 were determined with the test item at concentrations of 6.25, 12.5, and 25% in dimethyl formamide, respectively.
The test item was found to be a skin sensitizer. Since the S.I. was increased above the threshold of 3 even at the lowest tested concentration, an EC3 value could not be derived.