Reaction products of 2,4-bis(2-chlorophenyl)-5-(3,4-dimethoxy phenyl)-1H-imidazole and 2-(2-chlorophenyl)-4,5-diphenyl-1H-imidazole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (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:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Purity: not reported as such

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: between 21.9 and 23.2 grams
- Housing: In stainless steel, wire-mesh cages suspended above cage boards. Singly or in pairs during quarantine. Singly after assignment to groups, and during the dosing and resting phases of the study. After final weighing (test day 5) until sacrifice, animals were housed one group per plastic shoebox cage with appropriate bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): an approximate 12-hour light/dark cycle

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

0% (vehicle control), 5%, 10%, 25%, or 50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Evaluated, findings not reported
- Irritation: The test substance was dosed as a liquid and did not appear to have severe skin-irritating capability (pH of the highest concentration tested was 5).

MAIN STUDY         
Body Weight: Test days 0 and 5
Dosing: Test days 0-2
Days of Rest: Test days 3-4
Injection of Radioactivity: Test day 5
Removal of Lymph Nodes: At sacrifice (test day 5)
Disintegrations per minute (dpm) data: Test day 6

ANIMAL ASSIGNMENT AND TREATMENT
- Assignment to Groups: Prior to study start using a randomly generated, computer-based algorithm such that individual pretest body weights did not vary more than 20% of the group mean.
- Daily Animal Health Observations: At least once daily to detect moribundity and mortality.
- Clinical Observations: Prior to administartion of each dose and prior to sacrifice

TREATMENT PREPARATION AND ADMINISTRATION:
Twenty-five μL of vehicle control, test substance, or positive control (25% in vehicle control) were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 0-2). Test days 3-4 were days of rest followed by intravenous injection of 20 μCi of ³H-Thymidine per mouse on test day 5.
Approximately 5 hours after the injection, animals were sacrificed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8ºC overnight. On test day 6, the single cell suspensions were counted on a beta counter and reported as disintegrations per minute (dpm).

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE: A stimulation index (SI) was derived for each experimental group by dividing the mean disintegrations per minute (dpm) of each experimental group by the mean dpm of the vehicle control group. A stimulation index of greater than or equal to 3.0 together with consideration of dose response and, where appropriate, statistical significance were used in the determination of a positive response.

Positive control substance(s):

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

Statistics:

Significance was judged at p < 0.05 except for dpm data that were judged at p < 0.01. Lymph node dpm data were transformed to Log to obtain normality or homogenous variances.

Results and discussion

Positive control results:

A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.21

Test group / Remarks:

5%

Remarks on result:

other: mean dpm of 645.75

Results 2

Parameter:

SI

Value:

1.71

Test group / Remarks:

10%

Remarks on result:

other: mean dpm 911.75

Results 3

Parameter:

SI

Value:

1.39

Test group / Remarks:

25%

Remarks on result:

other: mean dpm 739.75

Results 4

Parameter:

SI

Value:

1.19

Test group / Remarks:

50%

Remarks on result:

other: mean dpm 632.75

Cellular proliferation data / Observations:

No statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at any test concentration. SIs of less than 3.0 were observed at all test concentrations of the test substance. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was not calculable.

Any other information on results incl. tables

Stimulation Index Data

GROUP	MATERIAL TESTED	n	MEAN (dpm)	S.D. (dpm)	SI
1	0% Vehicle Control	5	532.35	265.86	N/A
2	5%	5	645.75	211.46	1.21
3	10%	5	911.75	291.80	1.71
4	25%	5	739.75	317.74	1.39
5	50%	5	632.75	138.47	1.19
6	25% Positive Control	5	4653.95	2585.00	8.74

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Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not a dermal sensitizer in mice.

Executive summary:

The objective of this study was to evaluate the potential of the test substance to produce a dermal sensitization response in mice using the local lymph node assay (LLNA). Five groups of 5 female CBA/JHsd mice were dosed for 3 consecutive days with 0% (vehicle control), 5%, 10%, 25%, or 50% the test substance on both ears. Propylene glycol (PG) was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in PG as a positive control. On test day 5 of the assay, mice received ³H-thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears from the test substance groups was then evaluated and compared to the vehicle control group.

 

No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any test concentration. No clinical signs of toxicity were observed in the study.

 

No statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at any test concentration. Stimulation indices (SIs) of less than 3.0 were observed at all test concentrations of the test substance. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was not calculable. A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with the test substance. Under the conditions of this study, the test substance did not produce a dermal sensitization response in mice.

 

Based on these data, the test substance is not a dermal sensitizer in mice.