Ethyl 3-phenyloxirane-2-carboxylate

Endpoint summary

Description of key information

Skin sensitisation (OECD TG 429): Sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 November 2010 - 21 December 2010

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:

22 July 2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

(CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.

The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014 Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70 %, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

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

Concentration:

Test item at concentrations of 1, 2.5, 5, 10 or 25 % v/v in vehicle.

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using three mice, one per test item concentration. The mice were treated by daily application of 25 µL of the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for two consecutive days (Days 1 and 2). The surviving mice were similarly treated with the undiluted test item or the test item at a concentration of 25% v/v in acetone/olive oil 4:1 on the following day (Day 3). The mice were observed twice daily on Days 1 and 2. The surviving animals were observed twice on Day 3 and once on Day 4. The remaining mouse was observed on Days 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mouse on Day 6. The bodyweight of the mice that were humanely killed was recorded immediately prior to termination.

The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between Days 1 to 3 and Days 1 to 6. A mean ear thickness increase of equal to or greater than 25 % was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main Test
Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 25%, 10%, 5%, 2.5% or 1% v/v in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration according to the vehicle determination record. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.

The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, α Hexylcinnamaldehyde, at a concentration of 25 % v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
The vehicle control group and the positive control group served as common controls.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:80µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)

Positive control results:

The positive control item, α-Hexylcinnamaldehyde 85%, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

Key result

Parameter:

EC3

Remarks:

%

Value:

4.7

Key result

Parameter:

other: NOEC %

Value:

2.5

Key result

Parameter:

SI

Value:

1.59

Remarks on result:

other: 1%

Key result

Parameter:

SI

Value:

2.62

Remarks on result:

other: 2.5%

Key result

Parameter:

SI

Value:

3.05

Remarks on result:

other: 5%

Key result

Parameter:

SI

Value:

7.81

Remarks on result:

other: 10%

Key result

Parameter:

SI

Value:

14

Remarks on result:

other: 25%

Cellular proliferation data / Observations:

PRELIMINARY SCREENING TEST
The animals treated with the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1 were humanely killed, on Days 2 or 4, due to the occurrence of clinical signs of toxicity that exceeded the moderate severity limit set forth in the UK Home Office Project Licence. Signs of systemic toxicity noted were hunched posture and lethargy. Pilo-erection was also noted in the animal treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1. Bodyweight loss was also noted in both animals. Very slight erythema on both ears and excessive irritation, indicated by an approximate 40% increase in mean ear thickness, were noted on Day 4 in the animal treated with the undiluted test item.
No signs of systemic toxicity, visual local skin irritation or irritation indicated by a ≥25% increase in mean ear thickness were noted in the animal treated with the test item at a concentration of 25% v/v in acetone/olive oil 4:1.
Based on this information the dose levels selected for the main test were 25%, 10%, 5%, 2.5% and 1% v/v in acetone/olive oil 4:1.

EC3 CALCULATION
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 4.7%.

CLINICAL OBSERVATIONS:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS
Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Interpretation of results:

other: Sensitising 1B

Remarks:

According to EU CLP Regulation (EC) No. 1272/2008 and its amendments

Conclusions:

The SI values calculated for the substance concentrations 1, 2.5, 5, 10 and 25 % were 1.59, 2.62, 3.05, 7.81 and 14.0, respectively. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 4.7% w/v was calculated. A NOEC of 2.5% is derived. The test substance was considered to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the substance has been tested according to OECD TG 429 test guideline and GLP principles. At 1, 2.5, 5, 10 and 25% the substance showed SI values of 1.59, 2.62, 3.05, 7.81 and 14.0, respectively. Reliable negative and positive controls were included. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 4.7% w/v was calculated. A NOEC of 2.5% is derived. Based on the results, the substance was considered to be a sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitisation potential of the substance has been tested according to OECD TG 429 test guideline and GLP principles. At 1, 2.5, 5, 10 and 25% the substance showed SI values of 1.59, 2.62, 3.05, 7.81 and 14.0, respectively. Reliable negative and positive controls were included. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 4.7% w/v was calculated. A NOEC of 2.5% is derived.

Justification for classification or non-classification

Based on the results, the substance is considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labelled with H317: May cause an allergic skin reaction according to EU CLP (EC No. 1272/2008 and its amendments).