Octanoic acid, 2-phenoxyethyl ester

Endpoint summary

Description of key information

Skin sensitisation in vitro (OECD 429) = not sensitising

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:

10 Dec 2008 - 12 Jan 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2010

Deviations:

yes

Remarks:

ear thickness measurement in pre-screen test not performed

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

adopted in 2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: range: 20 - 24 g (main study); 28 - 30 g (preliminary study)
- Housing: during acclimatisation in groups, during test individually in Macrolon cages, containing sterilized sawdust as bedding material and paper as cage-enrichment (removed prior to dosing until Day 3)
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50 and 100% (undiluted) (v/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Compound was soluble in the vehicle and stable for over 96 h
- Irritation:
Two test substance concentrations were tested; a 50% and 100% (undiluted) concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids). The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study, unless otherwise specified. Two young adult animals were selected (8 - 14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3 - 4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.
Skin reaction:
animal 1 (50% test substance): erythema score = 1; edema score = 0
animal 2 (100% test substance): erythema score = 1; edema score = 0
Body weight:
animal 1 (50% test substance): Day 1 = 28 g; Day 3 = 27 g
animal 2 (100% test substance): Day 1 = 30 g; Day 3 = 28 g

Based on the results, the highest test substance concentration selected for the main study was a 100% (undiluted) concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine determined by beta-scintillation
- Criteria used to consider a positive response: Stimulation indices were calculated. The results were evaluated according to EC criteria for classification according to 67/548/EEC and EC 1272/2008.

TREATMENT PREPARATION AND ADMINISTRATION: 25 μl of 2-phenoxyethyl octanoate was applied to the entire dorsal surface of each ear of each mouse. The application was repeated on Days 2 and 3; local irritation reactions were assessed. On Day 6 an injection of 250 μl phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, animals were sacrificed and both ears were punched in the apical area. Punches were weighed and pooled per animal. In addition, the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of lymph node cells was prepared from each mouse by gentle separation through stainless steel gauze. Cells were washed twice with an excess of PBS and centrifuged at 200 g for 10 min at 4 °C. Macromolecules were precipitated with 5% trichloroacetic acid at 4 °C over night. Precipitated DNA was recovered, mixed with scintillation cocktail and the radioactivity was measured in a beta-counter.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated per group.

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX. In this study, performed in September 2008, females of the CBA mouse strain (from Charles River France, L‘Arbresle Cedex, France) were checked for the sensitivity to hexyl cinnamic aldehyde technical grade. The females were approximately 11 weeks old at the start of the study. Concentrations used for this study were 5, 10 and 25% in Acetone:Olive oil (4:1).

results: mean± SEM [DPM]; mean ± SEM [SI]
control: 472 ± 37; 1.0 ± 0.1
5%: 694 ± 235; 1.5 ± 0.5
10%: 901 ± 220; 1.9 ± 0.5
25%: 2879 ± 681; 6.1 ± 1.5

An EC3 value of 13.9% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6-monthly reliability checks of the recent years were 9.5, 13.1, 15.6, 14.1 and 13.8%.

Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing of contact hypersensitivity.

Key result

Parameter:

SI

Value:

1.3

Variability:

0.5

Test group / Remarks:

25% test substance

Key result

Parameter:

SI

Value:

1.3

Variability:

0.4

Test group / Remarks:

50% test substance

Key result

Parameter:

SI

Value:

1.8

Variability:

0.5

Test group / Remarks:

100% (undiluted) test substance

Key result

Parameter:

SI

Value:

6.1

Variability:

1.5

Test group / Remarks:

25% hexylcinnamicaldehyde

Cellular proliferation data / Observations:

EC3 CALCULATION
EC3 calculation could not be performed, as the SI values were < 3.0.

CLINICAL OBSERVATIONS
No clinical signs were observed and no mortality occured during the study period.

BODY WEIGHTS
The body weight development was normal for this species and strain.

IRRITATION
One or both ears of all animals at 50% and both ears of animals at 100% (undiluted) showed slight erythema (score 1). No edema was observed in any of the animals examined. Mean ear weight/animal for the experimental groups treated with test substance concentrations 25, 50 and 100% (undiluted) were 30.57, 31.51 and 31.16 mg, respectively. The mean ear weight/animal for the vehicle control group was 29.62 mg.

Table 1 Results of LLNA

Group	% test substance	DPM/animal	mean DPM± SEM	SI ± SEM
1	vehicle	406	646 ± 127	1.0 ± 0.3
		394
		592
		761
		1076
2	25	855	817 ± 270	1.3 ± 0.5
		227
		275
		1692
		1036
3	50	799	849 ± 156	1.3 ± 0.4
		994
		387
		1335
		731
4	100	1847	1193 ± 185	1.8 ± 0.5
		886
		1213
		793
		1225

DPM = disintegrations per minute

SEM = standard error of mean

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A local lymph node assay (LLNA) was performed with 2-phenoxyethyl octanoate according to OECD guideline 429 (Notox, 2009). A pre-screen test was conducted to select suitable doses. In the main study, 5 female CBA mice were treated with the test substance at 25, 50 and 100% (undiluted) on three consecutive days. One or both ears of all animals at 50% and both ears of all animals at 100% showed slight erythema (score 1). No oedema was observed in any of the animals examined. Based on ear weights, the observed slight irritation of the ears was considered not to have toxicologically significant influence on the activity of the lymph nodes. The lymph nodes of the animals in the experimental and control groups were considered to be normal in size. No mortality occurred and no symptoms of systemic toxicity were observed in the animals during the main study. Mean ear weight/animal for the experimental groups treated with test substance concentrations 25, 50 and 100% (undiluted) were 30.57, 31.51 and 31.16 mg, respectively. The mean ear weight/animal for the vehicle control group was 29.62 mg. The SI values calculated for the substance concentrations 25, 50 and 100% were 1.3, 1.3 and 1.8, respectively. The SI value of the positive control 25% hexyl cinnamic aldehyde in Acetone:Olive oil (4:1) was 6.1.

Based on the results of the conducted LLNA, 2-phenoxyethyl octanoate is considered to be not sensitizing to the skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available data on skin sensitisation with 2-phenoxyethyl octanoate does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.