Ethyl (S)-2-hydroxypropionate

Endpoint summary

Description of key information

The potential of the target substance ethyl (S)-lactate to induce skin sensitisation was tested in a suitable in vivo test method conducted according to OECD TG 429. Based on the results, ethyl (S)-lactate was not considered to be sensitising to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-03 to 2011-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

April 2002

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Trade name: Purasolv EL
- Batch number: 0909001195
- Appearance: liquid
- Purity: 99.83 %
- Storage: Room temperature, in the dark under nitrogen

Species:

mouse

Strain:

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 labelled 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 described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0
- Humidity (%): relative humidity of 40–70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark/hrs light): 12 / 12

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 interperitoneally 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 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)

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 of 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 to 20%.

Parameter:

SI

Value:

0.9

Variability:

+/- 0.4

Test group / Remarks:

25 % w/w

Parameter:

SI

Value:

1

Variability:

+/- 0.4

Test group / Remarks:

50 % w/w

Key result

Parameter:

SI

Value:

0.8

Variability:

+/- 0.4

Test group / Remarks:

100 % w/w

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

Since there was no indication that the test substance elicit an SI ≥ 3, no EC3 value could be calculated.

Cellular proliferation data / Observations:

Preliminary irritation study:
No irritation was observed in any of the animals examined.

Main study:
Skin reactions/irritation: No irritation of the ears was observed in any of the animals examined.
Macroscopy of the auricular lymph nodes and surrounding area: 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: 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.
Radioactivity measurements: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100 % were 651, 706 and 589 DPM respectively.The mean DPM/animal value for the vehicle control group was 735.
Toxicity and mortality: 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 substance^1(% 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 substance^1 (% 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).

Interpretation of results:

other: CLP criteria not met

Conclusions:

Under the given conditions, the test item was considered not to be a skin sensitiser.

Executive summary:

In a dermal sensitisation study conducted according to OECD TG 429 with ethyl (S)-lactate (purity: 99.83%) in acetone/olive oil (4:1 v/v), four groups of 5 female young adult CBA/J mice/dose group were tested in the local lymph node assay. The test item was administered epidermally to the dorsal surface of both ears, once a day on three consecutive days, at concentrations of 25, 50, or 100% (w/w). The volume administered was 25 µL per ear. A positive control (hexyl cinnamic aldehyde in acetone/olive oil (4:1 v/v)) was tested concurrently under identical conditions.

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 and no symptoms of systemic toxicity were observed in the animals. The SI values calculated for the substance concentrations of 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, no EC3 value could be calculated.

Based on these results, the test item was considered to be a non-skin sensitiser. Therefore, classification of ethyl (S)-lactate for skin sensitisation according to CLP Regulation 1272/2008 is not warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In a dermal sensitisation study conducted according to OECD TG 429 with ethyl (S)-lactate (purity 99.83%) in acetone/olive oil (4:1 v/v), four groups of 5 female young adult CBA/J mice/dose group were tested in the local lymph node assay. The test item was administered epidermally to the dorsal surface of both ears, once a day on three consecutive days, at concentrations of 25, 50, or 100% (w/w). The volume administered was 25 µL per ear. A positive control (hexyl cinnamic aldehyde in acetone/olive oil (4:1 v/v)) was tested concurrently under identical conditions.

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 and no symptoms of systemic toxicity were observed in the animals. The SI values calculated for the substance concentrations of 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, no EC3 value could be calculated.

Based on these results, the test item was considered to be a non-skin sensitiser. Therefore, classification of ethyl (S)-lactate for skin sensitisation according to CLP Regulation 1272/2008 is not warranted.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on available data, the test item can be considered as non-skin sensitiser. Therefore, classification of ethyl (S)-lactate for skin sensitisation according to CLP Regulation 1272/2008 is not warranted.