Ethyl 4-methyl-2-oxocyclohexanecarboxylate

Endpoint summary

Description of key information

A LLNA (OECD 429) study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α-Hexylcinnamaldehyde, 97.3%, at a concentration of 25% v/v in acetone/olive oil 4:1.

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 82%.

Teatment Group	Concentration	Stimulation Index	Result
Test Item	25% v/v in acetone/olive oil 4:1	1.18	Negative
	50% v/v in acetone/olive oil 4:1	1.66	Negative
	100%	3.75	Positive
Positive Control Item	25% v/v in acetone/olive oil 4:1	4.87	Positive

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:

The study was conducted between 28 October 2016 and 13 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 429 using the Skin Sensitisation: Local Lymph Node Assay method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Identification: FRET 13-0545
Physical state/Appearance: clear colorless liquid
Storage Conditions: approximately 4 °C in the dark

Species:

mouse

Strain:

other: CBA/Ca (CBA/Ca CruBR)

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/Ca CruBR) strain mice were supplied by Charles River UK Limited, Margate, Kent, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 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 8 to 12 weeks old.

The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 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:

other: acetone/olive oil 4:1

Concentration:

Undiluted test item the test item at concentrations of 50% or 25% v/v prepared as a solution in acetone/olive oil 4:1.

No. of animals per dose:

A preliminary screening test was carried out using a single mouse.
Groups of five mice were treated in the main test

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 one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), 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 time periods Days 1 and 3 and Days 1 and 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 sensitization.

Main Test
Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin 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, 97.3%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.


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 labeled with the study 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 10 minutes. The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 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 20 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. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were 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:

Dissiminated (delete when editing)
The positive control item, α-Hexylcinnamaldehyde, tech., 97.3%, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

Parameter:

SI

Value:

1.18

Test group / Remarks:

25% v/v

Remarks on result:

other: The following SI values were derived at 25, 50 and 100% v/v, respectively. Based on these results the EC3 is 82%.

Parameter:

SI

Value:

1.66

Test group / Remarks:

50% v/v

Parameter:

SI

Value:

3.75

Test group / Remarks:

100% v/v

Parameter:

other: disintegrations per minute (DPM)

Value:

880.8

Variability:

± 212.78

Test group / Remarks:

25% v/v

Remarks on result:

other: see Remark

Remarks:

Interpretation of Results The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser". The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is: EC3 = c + [[(3-d)/(b-d)] x (a-c)] where a = lowest concentration giving stimulation index >3 b = actual stimulation index caused by ‘a’ c = highest concentration failing to produce a stimulation index of 3 d = actual stimulation index caused by ‘c’. The radioactive disintegrations per minute per animal and the stimulation index were recorded. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are provided.

Parameter:

other: disintergrations per minute (DPM)

Value:

1 245.17

Variability:

± 221.13

Test group / Remarks:

50% v/v

Parameter:

other: disintergrations per minute (DPM)

Value:

2 809.55

Variability:

± 327.52

Test group / Remarks:

100% v/v

Preliminary test:

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1were selected for the main test.

Main test:

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

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Estimation of the Proliferative Response of Lymph Node Cells

Treatment Group	Concentration	Stimulation Index	Result
Test Item	25%v/vin acetone/olive oil4:1	1.18	Negative
	50%v/vin acetone/olive oil4:1	1.66	Negative
	100%	3.75	Positive
Positive Control Item	25%v/vin acetone/olive oil4:1	4.87	Positive

Interpretation of results:

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

Remarks:

Criteria used for interpretation of results: Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures and the Globally Harmonized System of Classification and Labelling of Chemicals

Conclusions:

The test item was considered to be a sensitiser under the conditions of the test and resulted in an EC3 of 82%, following OECD TG 429.

Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method. At 25, 50 and 100% the substance showed SI values of 1.18, 1.66 and 3.75 , respectively. An EC3 has been derived resulted in an EC3 of 82%.

The test item was considered to be a sensitizer under the conditions of the test.

The test item was also classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H317: May cause an allergic skin reaction” are therefore required.

The positive control α-Hexylcinnamaldehyde, 97.3% gave a Stimulation Index of greater than 3 (4.87) when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Migrated from Short description of key information:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method. At 25, 50 and 100% the substance showed SI values of 1.18, 1.66 and 3.75 , respectively. An EC3 has been derived resulted in an EC3 of 82%.

Justification for selection of skin sensitisation endpoint:
The result of this study is reliable and adequate for covering this endpoint.

Justification for classification or non-classification

Based on the EC3 value of 82% in the LLNA study, the substance has to be classified for skin sensitisation. According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, it has to be classified for skin sensitisation,Category 1B, H317: May cause an allergic skin reaction. According to EU Directive 67/548/EEC this results in: Xi-R43: May cause sensitisation by skin contact.