2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

EC Number:

263-471-3

EC Name:

2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Cas Number:

62256-00-2

Molecular formula:

C15H26O3

IUPAC Name:

2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate

Test material form:

liquid

Details on test material:

The test substance is identified as 2-ethylhexyl 7-oxabicyclo[4.1.0]heptane-3-carboxylate. The purity is 100%. The physical state/appearance of material is clear colorless liquid. The expiry date of material is 01 February 2019. The substance can be stored at 4 oC in dark conditions.

Specific details on test material used for the study:

Identification: 2-ethylhexyl 7 -oxabicyclo [ 4 .1. 0 ]heptane-3-carboxylate
Batch: AH03201
CAS Number: 62256-00-2
Purity: 100%
Physical state I Appearance: Clear colourless liquid
Expiry Date: 01 February 2019
Storage Conditions: Approximately 4 °C in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

Animal Information

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. 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.

Animal Care and Husbandry

The animals were housed in suspended solid floor polypropylene cages furnished with softwood wood flakes. 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.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

50 or 25%

No. of animals per dose:

4

Details on study design:

Test Item Administration
Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil4: 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 11L 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 four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (250 11L) of phosphate buffered saline (PBS) containing 3H-methyl thymidine eHTdR: 80 11Ci/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 11Ci 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. Body Weights: The body weight 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. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 cell 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 pooled 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. 3HT dR incorporation was measured by 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:

The proliferation response of lymph node cells was expressed as the number of radioactive
disintegrations per minute per lymph node (disintegrations per minute/node) 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 sensitizer 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-sensitizer".

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)]

Major Computerized Systems
The following computerized system was used in the study:
Delta Controls- ORCA view

Results and discussion

Positive control results:

Acceptible results were produced in the periodic positive control tests

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Value:

33.6

Results 2

Parameter:

SI

Value:

2.29

Test group / Remarks:

25%

Results 3

Parameter:

SI

Value:

4.36

Test group / Remarks:

50%

Results 4

Parameter:

SI

Value:

7.64

Test group / Remarks:

100%

Any other information on results incl. tables

Tables were not able to be pasted from a .PDF successfully, so they are attached as a single document attachment

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

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

Executive summary:

Introduction

A 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.

Methods

Following a preliminary  screening test in which no clinical signs of toxicity were noted at a concentration  of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.  Three groups, each of four animals, were treated with 50 ul (25 ul 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 four animals was treated with acetone/olive  oil 4:1 alone.

Results

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 as follows:

Concentration (% v/v) in              Stimulation Index              Result

acetone/olive oi (4:1)

25                                                        2.29                     Negative

50                                                        4.36                     Positive

100                                                      7.64                     Positive

The concentration  of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 33.6%.

Conclusion

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