Oxirane, 2,2'-[methylenebis[(2,6-dimethyl-4,1-phenylene)oxymethylene]]bis-

Endpoint summary

Description of key information

Skin sensitisation

The test material was considered to be a sensitiser under the conditions of the test.

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:

19 June 2006 to 11 July 2006

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 15 - 23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 30 - 70 %
- Air changes: approximately fifteen changes per hour
- Photoperiod: twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

Vehicle:

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

Concentration:

10 %, 25 % or 50 % w/w in acetone/olive oil 4:1

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
A single mouse was treated by daily application of 25 µL of the test material at a concentration of 50 % w/w in acetone/olive oil 4:1, 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. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.

MAIN STUDY
- Test Material Administration
Groups of four mice were treated with the test material at concentrations of 10 %, 25 % or 50 % w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test material 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 material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material 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 material (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol, ) 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 illhealth 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. 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 labelled with the project 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 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 measurd 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 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.

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

A test material is regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation is classified as a "non-sensitiser".

Positive control substance(s):

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

Positive control results:

Tested concentrations of positive control in butanone of 5, 10 and 25 % v/v produced Stimulation Indeces of 3.08, 4.54 and 8.06, repectively. The positive control was therefore considered to be a sensitiser under the conditions of the test.

Key result

Parameter:

SI

Value:

5.57

Test group / Remarks:

10 % w/w in vehicle

Key result

Parameter:

SI

Value:

8.67

Test group / Remarks:

25 % w/w in vehicle

Key result

Parameter:

SI

Value:

4.98

Test group / Remarks:

50 % w/w in vehicle

Cellular proliferation data / Observations:

- Estimation of the Proliferative Response of Lymph Node Cells
A stimulation index of greater than 3 was recorded for the three concentrations of the test material (10 %, 25 % and 50 % w/w in acetone/olive oil 4:1).

- Clinical Observations and Mortality Data
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. White residual test material was noted post dose on the ears of animals treated with the test material at a concentration of 50 % w/w in acetone/olive oil 4:1 on Days 1, 2 and 3, on three animals on Days 4 and on two animals on Day 5.

- Bodyweight
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.

Preliminary Screening Test

No signs of systemic toxicity were noted. White residual test material was noted on the ears post dose on Days 2 and 3.

Based on this information the dose levels selected for the main test were 10 %, 25 % and 50 % w/w in acetone/olive oil 4:1.

Table 1: Main Test Results - DPM, DPM/node and Stimulation Index

Concentration (% w/w) in vehicle	DPM	DPM/node	Stimulation Index	Result
Vehicle	4156.65	519.58	N/A	N/A
10	23170.71	2896.34	5.57	Positive
25	36030.94	4503.87	8.67	Positive
50	20706.81	2588.35	4.98	Positive

Interpretation of results:

other: Skin sensitiser according to EU criteria

Conclusions:

The test material was considered to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitising potential of the test material was investiaged in a study which was conducted in accordance with the standardised guidelines OECD 429 and EU Method B.42, under GLP conditions.

Following a preliminary screening test, three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations of 10 %, 25 % or 50 % w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was 5.57, 8.67 and 4.98 at concentrations of test material in acetone/ olive oil 4:1 of 10, 25 and 50 % w/w, respectively.

The test material was therefore considered to be a sensitiser under the conditions of the test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation

The skin sensitising potential of the test material was investiaged in a study which was conducted in accordance with the standardised guidelines OECD 429 and EU Method B.42, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Following a preliminary screening test, three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations of 10 %, 25 % or 50 % w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was 5.57, 8.67 and 4.98 at concentrations of test material in acetone/ olive oil 4:1 of 10, 25 and 50 % w/w, respectively.

The test material was therefore considered to be a sensitiser under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does require classification with respect to skin sensitisation (category 1) and is assigned the hazard statement H317: May cause an allergic skin reaction.