Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch no.: 0909001195
Purity: 99.83 w/w %

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, 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 (20 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. Accomodation was as desribed 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: 20.1 - 23.3°C).
- Humidity (%): relative humidity of 40-70% (actual range: 37-62%).
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50 and 100% w/w.
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Irritation: No irritation of the ears was observed in any of the animals examined. Based on the results, the highest test substance concentration selected for the main study was a 100% concentration.


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 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. 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. No adjustment was made for specific gravity of the vehicle or density of the test substance. 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 irritation of 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 radioacticity - 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 200g 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, resuspenden i 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 1.3, 1.7 and 4.1 respectively. An EC3 value of 18.1% was calculated using linear interpolation.The calculated EC3 value was found to be in the acceptable range of 2 and 20%.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100% were 0.9, 1.0 and 0.8, respectively. Since there was no indication that the test substance elicit an SI ≥ 3, no EC3 value could be calculated See Table 2 below.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 651, 706 and 589 DPM respectively. See Table 1 and 2 below. The mean DPM/animal value for the vehicle control group was 735 DPM.

Any other information on results incl. tables

No irritation of the ears was observed in any of the animals examined. 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.

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.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Table 1: Radioactivity measurements (individual animals)

group

Test substance1(%w/w)

animal

DPM/animal

 

 

 

 

1

0%

(vehicle)

1

400

2

646

3

778

4

163

5

1688

 

 

 

 

2

25%

6

449

7

356

8

564

9

776

10

1110

 

 

 

 

3

50%

11

549

12

872

13

807

14

776

15

527

 

 

 

 

4

100%

16

616

17

188

18

1365

19

306

20

471

1Vehicle: Acetone/Olive oil (4:1 v/v).

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

group

test substance1

(% w/w)

median

DPM

SI

2

25%

651 ± 134

0.9 ± 0.4

3

50%

706 ± 70

1.0 ± 0.4

4

100%

589 ± 207

0.8 ± 0.4

 

 

 

 

1

0% (vehicle)

735 ± 261

1.0 ± 0.5

1Vehicle: Acetone/Olive oil (4:1 v/v).

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
PURASOLV®EL (ethyl-S-lactate) would not be regarded as skin sensitizer according to the recommendations made in the test guidelines and does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemical (GHS) of the United Nations (2007) and the Regulation (EC) No1272/2008 on classification, labelling and packaging of substances and mixtures.
Executive summary:

In a dermal senisitization study with PURASOLV®EL (99.83% a.i.) (ethyl-S-lactate) in Acetone/Olive oil (4:1 v/v), young adult CBA/J mice (5 females/group) were tested (0, 25, 50 and 100% substance concentration) using the method of LLNA. As positive control material Alpha-hexylcinnamicaldehyde was used. No irritation of the ears was observed in any of the animals examined. 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 an no symptoms of systemic toxicity were observed in the animals.

The SI values calculated for the substance concentrations 25, 50 and 100% were 0.9, 1.0 and 0.8 respectively. Since there was no indication that the test substance elicits an SI≥ 3 when tested up to 100%, PURASOLV®EL (ethyl-S-lactate) was considered to be a non skin sensitizer.

In this study, PURASOLV®EL (ethyl-S-lactate) is no skin sensitizer.