1,4-Dioxane-2,5-dione, 3,6-dimethyl-, (3R,6R)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-06-07 to 2010-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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 April 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(3S-cis)-3,6-dimethyl-1,4-dioxane-2,5-dione

EC Number:

224-832-0

EC Name:

(3S-cis)-3,6-dimethyl-1,4-dioxane-2,5-dione

Cas Number:

4511-42-6

Molecular formula:

C6H8O4

IUPAC Name:

3,6-dimethyl-1,4-dioxane-2,5-dione

Specific details on test material used for the study:

- Name of test material (as cited in study report): L-Lactide
- Expiration date of the lot/batch: 2010-11-23
- Stability under test conditions: stable
- Appeatance: white flakes
- Storage condition of test material: in refrigerator (2-8 °C) in the dark under nitrogen
- Analytical purity: 99%
- Lot/batch No.: 0911001952

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: young adult animals (approximately 10 weeks old).
- Weight at study initiation: body weight variation was within ± 20% of the sex mean (23 gram).
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on day 1 prior to dosing and was supplied again after scoring of the ears on day 3.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF@Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of the treatment under laboratory conditions. Accommodation was as desrcibed above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 °C (actual range: 20.9-23.0 °C).
- Humidity (%): relative humidity of 40-70 % (actual range: 42-81 %).
- Air changes (per h): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From 07 June 2010 to 12 July 2010

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10, 25 and 50% w/w

No. of animals per dose:

5 females per dose group

Details on study design:

RANGE FINDING TESTS:
A preliminary irritation study was conducted. Two test substance concentrations were tested: 25 and 50%. The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study. Two young adult animals were selected. Each animal was treated with one concentration on three consecutive days. Ca 3-4 hours after the last exposure, the irrotation of the ears was assessed. Bodyweights were determined on day 3.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA.
Animal assignment: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality. No further information of animal assignment is available.
- Criteria used to consider a positive response: DPM values are presented for each animal and each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.

Induction - days 1,2 and 3:
The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that the vehicle was administered instead of the test substance. Approximately 3-4 hours after the last exposure, the irritationof the ears was assessed.
Excision of the nodes - day 6:
All animals: Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of ³H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After approximately five hours, all animals were killed by interperitoneal injection with Euthasol® 20 % (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 ml PBS.

Tissue processing for radioactivity - day 6:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gently separation through stainless steel gauze (diameter 125 µm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) stored in the refrigerator until the next day.

Radioactivity measurements - day 7:
Precipitates were recovered by centrifugation, resuspended in 1 ml TCA and transferred to 10 ml of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2 % or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert counts per minute (CPM) to disintegrations per minute (DPM).

Positive control substance(s):

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

Results and discussion

Positive control results:

The six-month reliability check with alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
The SI values calculated for alpha-hexylcinnamicaldehyde concentrations 5, 10 and 25% were 1.7, 2.7 and 8.8 respectively. An EC3 value of 10.7% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.1

Test group / Remarks:

low dose (10% test substance)

Results 2

Parameter:

SI

Value:

1.5

Test group / Remarks:

mid-dose (25% test substance)

Results 3

Parameter:

SI

Value:

0.9

Test group / Remarks:

high-dose (50% test substance)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION:
The SI values calculated for the substance concentrations 10, 25 and 50% were 2.1, 1.5 and 0.9, respectively. Since there was no indication that the test substance elicit an SI ≥ 3 when tested up to 50%, L-lactide was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI=3) (if any) exceeds 50%. See Table 2 below.

CLINICAL OBSERVATIONS:
No irritation of the ears was observed in any of the animals examined. White remnants of the test substance were present on the ears at the 50% concentration, but this did not hamper the scoring of erythema. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Red discolouration of the left auricular lymph node was noted for one male (no. 12) at 25%. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

BODY WEIGHTS:
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness):
No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Any other information on results incl. tables

Table 1: Radioactivity measurements (individual animals)

group	Test substance^1(% w/w)	animal	DPM/animal
1	0% (vehicle)	1	481
		2	378
		3	391
		4	273
		5	379
2	10%	6	681
		7	1173
		8	381
		9	786
		10	914
3	25%	11	524
		12	366
		13	842
		14	707
		15	405
4	50%	16	324
		17	207
		18	426
		19	501
		20	211

¹Vehicle: Dimethyl formamide.

Table 2: Disintegration Per Minute (DPM) and Stimulation Index (SI)

group	test substance^1 (% w/w)	median DPM ± SEM	SI ± SEM
2	10%	787 ± 131	2.1 ± 0.4
3	25%	569 ± 90	1.5 ± 0.3
4	50%	334 ± 58	0.9 ± 0.2
1	0% (vehicle)	380 ± 33	1.0 ± 0.1

¹Vehicle: Dimethyl formamide.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in a mouse local lymph node assay, L-lactide is considered not to be a skin sensitizer.

Executive summary:

In a dermal senisitization study with L-lactide (purity 99%) in dimethyl formamide, young adult CBA/J mice (5 females/group) were tested (0, 10, 25 and 50% substance concentration) according to OECD 429 (LLNA). As positive control material alpha-hexylcinnamicaldehyde was used. No irritation of the ears was observed in any of the animals examined. Red discouloration of the left auricular lymph node was noted for one male in the 25% treatment group. All 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. No mortality occurred and no symptoms of systemic toxicity were observed in the animals.

The SI values calculated for the substance concentrations 10, 25 and 50% were 2.1, 1.5 and 0.9 respectively. Since there was no indication that the test substance elicits an SI≥ 3 when tested up to 50%, L-lactide was considered not to be a skin sensitizer.