N,N”-hexane-1,6-diylbis(N’-alkylaryl)urea

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 - 29 Sep 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

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:

- Name of test material (as cited in study report): trade name given
- Substance type: white powder
- Analytical purity: 99.5%
- Lot/batch No.: 1592ZG-076
- Expiration date of the lot/batch: Aug 2015
- Storage condition of test material: at room temperature
- Other: pH: 6.4-6.3 (1% in water)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J strain, inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: Pre-screen test: 20-24 g, Main test: 19-25 g
- Housing: group housed in Makrolon cages (MIII type; height 18 cm), sterilized sawdust was used as bedding material, paper and shelters as cage-enrichment
- Diet: pelleted rodent diet, SM R/M-Z (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Sep 2014 To: 29 Sep 2014

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

5, 10 and 25% (w/w)

No. of animals per dose:

range finding study: 2 females
main study: 5 females

Details on study design:

RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at most (maximum grade 2 and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied. Two test substance concentrations (10% and 25%) were tested. 25% was the highest concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.
No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. White staining of the dorsal surface of the ears by test substance remnants was noted for all animals (10% Days 1-3; 25% Days 1-4). Based on these results, the highest test substance concentration selected for the main study was 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A Stimulation Index (SI) was calculated for each group using the individual SI values. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The application was repeated on Days 2 and 3; local irritation reactions were assessed. On Day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid and stored in the refrigerator until the next day.

Positive control substance(s):

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

Results and discussion

Positive control results:

The results of a reliability test with three concentrations of hexyl cinnamic aldehyde in acetone/olive oil (4:1 v/v) was performed not more than 6 months previously (Apr 2014) using the same materials, animal supplier, animal strain and essential procedures. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.4 and 4.7 respectively. An EC3 value of 17.3% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 16.9, 14.4, 16.5, 14.5, 13.4 and 14.1%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.2 and 1.3 respectively. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 788, 843 and 865 DPM respectively. The mean DPM/animal value for the vehicle control group was 684 DPM.

Any other information on results incl. tables

Table1: Relative size lymph nodes, radioactivity counts (DPM) and Stimulation Index (SI).

Group	TS^1(%)	Animal	Size nodes^2		DPM^3/aninmal	Mean DPM±SEM^4	Mean SI±SEM
			left	right
1	0	1	n	n	863	684 ± 129	1.0 ± 0.3
		2	n	n	191
		3	n	n	915
		4	n	n	764
		5	n	n	688
2	5	6	n	n	1117	788 ± 98	1.2 ± 0.3
		7	n	n	725
		8	n	n	782
		9	n	n	504
		10	n	n	810
3	10	11	n	n	521	843 ± 123	1.2 ± 0.3
		12	n	n	1144
		13	n	n	1090
		14	n	n	838
		15	n	n	621
4	25	16	n	n	1128	865 ± 93	1.3 ± 0.3
		17	n	n	722
		18	n	n	749
		19	n	n	860
		20^5	n

¹TS = test substance (% w/w)

²Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal).

³DPM= Disintegrations per minute

⁴SEM = Standard Error of the Mean

⁵Right node was not found during section. The DPM value for this animal was not used for interpretation since this value is based on one node only. Based on the available data, it was considered that the study outcome was not adversely affected.

Skin reactions: No irritation of the ears was observed in any of the animals examined. White staining of the dorsal surface of the ears by test substance remnants was noted for all experimental animals between Days 1 and 3. The staining did not hamper the scoring of the ears.

Systemic toxicity and body weights: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopy of the auricular lymph nodes and surrounding area: The auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified