Sodium 4-(4-substitutedanilino)-4-substituedbut-2-enoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 July - 15 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

(individual housing of mice instead of group housing due to aggression in caged mice)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

(individual housing of mice instead of group housing due to aggression in caged mice)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in a cold dark place (prefabricated refrigerator, 8.6 to 11.4°C), protected from light, in a well-closed container.
- Stability of test article: stable for one year, at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/J [SPF]

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Japan
- Age at study initiation: 9 weeks
- Weight at study initiation: 20.5 - 24.6 g
- Housing: individual in wire mesh metal cages with an automatic water flushing breeding rack (Toyoriko), the feeders were exchanged once a week
- Diet: pellet diet CRF-1 (Oriental Yeast, lot No. 131108), ad libitum
- Water: ad libitum (analysis was performed)
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.9 - 23.1
- Humidity (%): 53.6 - 62.2
- Air changes (per hr): ≥ 12
- Photoperiod (hrs dark / hrs light): 12/ 12
- IN-LIFE DATES: From: 09 July To: 15 July 2014

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

(DMF)

Concentration:

10, 25 and 50% (w/v) in DMF; prepared just before use

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TEST:
In the pre-screening test, three concentrations (10, 25 and 50% solution in DMF) were selected, and 25 μl each dose formulation were applied on the dorsal skin of both auricles of each animal, 2 mice for each concentration, once a day for 3 consecutive days. The general conditions including an observation of the application site were performed once each day (from 1 to 3 hours after the application to day 6). As a result, no animals showed any abnormalities. The body weights and ear thickness were measured before the initial application and on Day 6. None of the animals showed any changes deviating from the criteria of body weights (less than 5% weight change) and ear thickness (less than 25% increase in ear thickness). No rationale given for dose selection in the pre-screen test.

From the results mentioned above, 50% was selected as the high concentration for the main study, because it was expected not to induce any toxic signs in the general condition, 25% or more increase in the ear thickness, dermal erythema with score of 3 or more on the auricles, or more than 5% of body weight loss, and two lower concentrations of 25 and 10%.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methylthymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A substance is regarded as a sensitizer in the LLNA, if the Stimulation Index (SI) is 3 or greater.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µl of each dose formulation were applied to the dorsal skin of auricles of each animal once a day (using a MICROMAN (Model M100, Gilson) for 3 days. On day 6 20 μCi [methyl-3H] thymidine (3HTdR) (= 250 μL of 80 μCi/mL 3HTdR solution) was administered intravenously to each mouse via the tail vein with disposable syringes and 27G needles. 5 h after administration local lymph nodes were collected and minced and then the pooled lymph node cells (LNC) were treated with 5% TCA overnight (approximately 18 hours) before determination of the ammount of 3HTdR incorporation on day 7.

Positive control substance(s):

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

Statistics:

No statistical analysis were performed.

Results and discussion

Positive control results:

The positive control substance (25% α-Hexylcinnamaldehyde (Lot # LAP0946, Wako Pure Chemical Industries, Ltd, Japan) in DMF) induced a positive reaction, determined by a DPM/animal of 5317.9 compared to 1094.5 DPM/animal in the control group, leading to a SI of 4.9. No abnormal clinical signs, erythema on the auricles or body weight changes on day 6 were observed. The weights of lymph nodes were however much larger than those in the vehicle control group (9.9 +/- 0.8 vs 5.2 +/- 0.8 in HCA treated animals and control group, respectively) and the ear thickness of animals treated with the test substance was larger than that in the vehicle control group (day 3: 0.15 +/- 0.009 mm vs 0.13 +/- 0.005; day 6: 0.18 +/- 0.009 mm vs 0.13 +/- 0.005, respectively).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.7

Test group / Remarks:

10 % test group

Results 2

Key result

Parameter:

SI

Value:

4

Test group / Remarks:

25% test group

Results 3

Key result

Parameter:

SI

Value:

3.1

Test group / Remarks:

50% test group

Results 4

Parameter:

SI

Value:

4.9

Test group / Remarks:

positive control group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Lymph node weight was not altered compared to that of the vehicle control group (5.2 +/- 0.8, 5.4 +/- 0.4 , 6.4 ± 0.8, 6.1 ± 0.7 for control, 10, 25 and 50% test substance, respectively). Topical application of the test substance led to an increase in the mean DPM values of pooled lymph nodes. The following values were obtained: 1094.5, 1831.8, 3432.9 and 3402.8 DPM/animal in control, 10, 25 and 50% test groups.

DETAILS ON STIMULATION INDEX CALCULATION
The SI was derived by dividing the mean DPM/mouse within each test substance group and positive control group by the mean DPM/mouse for the vehicle control group. The following values were obtained: 1.7, 4.0, 3.1 and 4.9 for 10, 25 and 50% test substance and the positive control substance HCA, respectively.

EC3 CALCULATION
The EC3 value was estimated via quadratic regression. The allergenic potency class of the test substance was assigned depending on the EC3 value using the GHS categorization scheme.
The EC3 value of the test substance was calculated to be 16.8% via quadratic regression.

CLINICAL OBSERVATIONS and BODY WEIGHTS
No abnormal clinical signs, erythema on the auricles or body weight increases or decreases on day 6 were observed. Neither individual ear thickness was altered compared to that of the vehicle control croup.

Any other information on results incl. tables

Table 1: Stimulation index in mice after application of the vehicle (DMF), test substance (10, 25, 50% in DMF) or positive control substance (25% HCA in DMF)

Compound	Concentration [%]	Number of animal	DPM/ animal	Stimulation index	Judgement	GHS classification
DMF	100	4	1094.5	-	-	-
Test substance	10	4	1831.8	1.7	Negative	1B
	25	4	4342.9	4.0	Positive
	50	4	3402.8	3.1	Positive
HCA	25	4	5317.9	4.9	Positive	-

 - = Not applicable

Stimulation index (SI) =DPM/animal of group 2 - 5)/(DPM/animal of group 1

Judgement: The cases showing three or greater SI values were defined as positive.

DMF = N,N -Dimethylformamide

HCA = α-Hexylcinnamaldehyde

The classification of skin sensitization was conformed to the "Globally Harmonized System of Classification and Labelling of Chemicals (GHS)" (5th revised edition, United Nations, 2013).

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

CLP: Skin Sens 1B, H317