Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-

Skin sensitisation

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:

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:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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:

Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-

EC Number:

606-097-1

Cas Number:

186817-80-1

Molecular formula:

C11H22O3

IUPAC Name:

Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-

Constituent 2

Reference substance name:

2-ethylhexyl-S-lactate

IUPAC Name:

2-ethylhexyl-S-lactate

Details on test material:

Batch no.: 0805000655
Purity: 99 %

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Purac Biochem, batch no. 0805000655
- Expiration date of the lot/batch: 05 September 2010
- Purity test date: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark under nitrogen
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No reactivity known

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None - not necessary

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Not applicable
- other information: –

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan, Horst, The Netherlands.
- 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 (21 grams).
- 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 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, 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)

Statistics:

Since outliers are present in this study, median DPM/animal values were used to interpret the data.
The EC3 value (the estimated test substance concentration that will give a SI = 3) was determined by linear interpolation.

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 0.7, 1.5 and 6.3 respectively. An EC3 value of 14.7 % was calculated by 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:

ca. 2.2

Test group / Remarks:

2 (25 % test substance)

Results 2

Parameter:

SI

Value:

ca. 2.1

Test group / Remarks:

3 (50 % test substance)

Results 3

Parameter:

SI

Value:

ca. 3.5

Test group / Remarks:

4 (100 % test substance)

Results 4

Parameter:

EC3

Remarks:

[% test substance]

Value:

ca. 82.1

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 animals at 50 % and 100 % were considered enlarged and all auricular lymph nodes of animals at 25 % and control animals 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 substance^1(%w/w)	animal	DPM/animal
1	0% (vehicle)	1	657
		2	605
		3	0^2
		4	1636
		5	660
2	25%	6	728
		7	1444
		8	4485
		9	671
		10	2371
3	50%	11	1020
		12	1369
		13	1127
		14	1380
		15	1964
4	100%	16	2280
		17	1955
		18	2815
		19	2849
		20	1865

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

²Value of animal no. 3 rejected and not used for interpretation (technical error).

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

group	test substance^1 (% w/w)	median DPM	SI
2	25%	1444	2.2
3	50%	1369	2.1
4	100%	2280	3.5
1	0% (vehicle)	659	1.0

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

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Based on the results from an LLNA (OECD 429) the test substance must be considered as skin sensitizer (Skin Sens 1B, H317).

Executive summary:

In a dermal senisitization study with PURASOLV®EHL (99%) (2-ethylhexyl-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 animals at 50 % and 100 % were considered enlarged and all auricular lymph nodes of animals at 25 % and control animals 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 PURASOLV®EHL (2-ethylhexyl-S-lactate) concentrations 25, 50 and 100 % were 2.2, 2.1 and 3.5, respectively. These results indicate that the test substance could elicit an SI ≥ 3. An EC3 value (the estimated test substance concentration that will give a SI = 3) of 82.1 % was calculated.

In this study, PURASOLV®EHL (2-ethylhexyl-S-lactate) is identified as a skin sensitiser (Skin Sens 1B, H317)