2-ethylhexyl lactate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

March-June 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study; test substance was 2-ethylhexyl-S-lactate, hence read-across to the non-stereospecific 2-ethylhexyl lactate (unspecified stereochemistry) is feasible without restrictions.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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)

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 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.

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 using linear interpolation.

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 using linear interpolation.The calculated EC3 value was found to be in the acceptable range of 2 and 20 %.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The SI values calculated for the substance 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. See Table 2 below.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Since outliers are present in this study, median values were used to interpret the data. Median DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100 % were 1444, 1369 and 2280 DPM respectively. See Table 1 and 2 below. The median DPM/animal value for the vehicle control group was 659 DPM.

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).

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

According to the recommendations made in the test guidelines, PURASOLV®EHL (ethylhexyl-S-lactate) would be regarded as skin sensitizer.
According to the Globally Harmonized System of Classification and Labelling of Chemical (GHS) of the United Nations (2007), PURASOLV®EHL (ethylhexyl-S-lactate) should be classified as skin sensitizer (Category 1).
According to the Regulation (EC) No1272/2008 on classification, labelling and packaging of substances and mixtures, 2-ethylhexyl lactate should be classified (by read-across from 2-ethylhexyl-S-lactate) as skin sensitiser (Category 1) and labeled as H317: May cause an allergic skin reaction.

Executive summary:

In a dermal senisitization study with PURASOLV®EHL (99 % a.i.) (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 2-ethylhexyl-S-lactate (hence also 2-ethylhexyl lactate, by read-across) is a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A dermal sensitisation study (LLNA) was performed with 99 % 2-ethylhexyl-S-lactate in acetone/olive oil (4:1 v/v).

The SI values calculated for the 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. The potency is not high; it falls within the lowest potency class (EC3 value > 2 % w/v, moderate potency).

Although the potency is low, the positive result in the test means that 2-ethylhexyl lactate (by read-across from 2-ethylhexyl-S-lactate) has to be classified as a skin sensitiser.

Migrated from Short description of key information:
2-Ethylhexyl-S-lactate gave a positive test result in the mouse local lymph node assay (LLNA). An EC3 value (the estimated test substance concentration that will give a SI = 3) of 82.1 % was calculated.

Justification for classification or non-classification

Results from an LLNA study with 2 -ethylhexyl-S-lactate 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. The potency is not high; it falls within the lowest potency class (EC3 value > 2% w/v, moderate potency).

Although the potency is low, the positive result in the test means that PURASOLV®EHL (2-ethylhexyl-S-lactate) has to be classified as Skin sensitisation: Category 1.