2-Oxazolidinone, 3-ethenyl-5-methyl-

Endpoint summary

Description of key information

5-Methyl-3-vinyloxazolidin-2-one showed no skin sensitizing potential in a Local Lymph Node Assay according to OECD guideline 429.

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:

18 March 2015 - 13 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 1st pre-test 10 – 11 weeks old; 2nd pre-test 8 - 9 weeks; 3rd pre-test 9 - 10 weeks; Main study: 9 - 10 weeks
- Housing: group housing Makrolon Type II (pre-test) Makrolon Type III cages (main study), with w ire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Relative Humidity (%): approx. 45 - 65; except for serveral hours
The relative humidity in the animal room was between approximately 36 – 65 % instead of 45 – 65% for few hours. This deviation to the study plan,
however, does not affect the validity of the study.
- Photoperiod (hrs dark / hrs light):artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

propylene glycol

Concentration:

0,5%, 1% , 2%

No. of animals per dose:

2 females for each pre-test ; 5 females per group (main-study)

Details on study design:

RANGE FINDING TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be
technically used was 100% of the undiluted test item. Test item solution at different concentrations was prepared using PG as vehicle.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in
two animals.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily
each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were
recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin.
Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a
micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm
corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered
to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.

On day 2 and 3, both animals showed an erythema of the ear skin (score 1, see Appendix 1 for details). At the tested concentrations both animals
showed signs of systemic toxicity such as hunched back, tippy toe walk, and increased spontaneous activity. Furthermore, a weight loss of >5% was observed in the animal treated with the undiluted test item during the course of the study.
Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. The animal treated with 25% showed an erythema of the
ear skin (score 1, see Appendix 1 for details) while both animals showed symptoms of systemic toxicity such as hunched back and ruffled fur. In the
animal treated with 10% a weight loss > 5% was observed both animals showed symptoms of toxicity such as hunched back and ruffled fur, as well as
some weight loss.
In the third pre-test concentration of 2 and 5% were used. Both animals showed an erythema of the ear skin (score 1, see Appendix 1 for details). The animal treated with 5% test item concentration showed signs of systemic toxicity such as reduced spontaneous activity, ruffled fur, and was chubby
cheeked, as well as some body weight loss >5% during the course of the study.
Thus, the test item in the main study was assayed at 0.5, 1, and 2%. The highest concentration tested was the highest level that could be achieved
whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Furthermore, an index was calculated for the lymph node weight and –cell count as well as for the ear weight by dividing the mean values of the test
item treated groups by the mean of the vehicle treated group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was
reported for a positive response and the cut-off value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1.
However, these cut-off values mentioned in the respective papers have been determined using a different strain of mice and can thus not be
implicitly adopted.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and PG was added. The different test item concentrations were prepared
individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 0.5, 1 and
2% in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear once daily for three
consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.5 µCi of ³H-methyl thymidine (equivalent to
81.8 µCi/mL ³HTdR) were injected into each test and control mouse via the tail vein.

DETERMINATION OF INCORPORATED ³HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes,
followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single
cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless
steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in
5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The
precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and
thoroughly mixed. The level of ³HTdR incorporation was then measured in a ß-scintillation counter. Similarly, background ³HTdR levels were also
measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses ³HTdR incorporation as the number of radioactive
disintegrations per minute (DPM).

DETERMINATION OF LYMPH NODE WEIGHT AND CELL COUNT:
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell
count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed.
Subsequently, individual cell counts were determined using a cell counter (CASY DT, Schärfe System). The values obtained were taken down manually.

DETERMINATION OF EAR WEIGHTS:
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm
corresponding to 0.5 cm²). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node
cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations validated statistical program R Script Decision Tree_2-Rnw was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program R Script
Outlier.Rnw).
However, both biological and statistical significance were considered together.

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control
experiment was performed using CBA/CaOlaHsd mice in April 2015

Parameter:

SI

Remarks on result:

other: In this study Stimulation Indices (S.I.) of 2.09, 2.77, and 2.40 were determined with the test item at concentrations of 0.5, 1 and 2 % in PG. The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant but biologically not relevant increase was observed for the lymph node cell count for both the low and the high dose groups in comparison to the vehicle control group. A statistically significant or biologically relevant increase in lymph node weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any dose group.

Calculation of Stimulation Indices per Dose Group:

Test item concentration	Group Calculation	SD	S.I.
	Mean DPM per animal (2 lymph nodes)^a)
Vehicle Control (PG)	472.7	208.5	1.00
0.5% 5 -Methyl-3 -vinyloxazolidin-2 -on	986.1	226.9	2.09
1% 5 -Methyl-3 -vinyloxazolidin-2 -on	1307.7	582.3	2.77*
2% 5 -Methyl-3 -vinyloxazolidin-2 -on	1132.7	521.8	2.40

a)  Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within

a group by the number of animals in that group (5 animals)

*   statistically significant (p<0.05).

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No signs of systemic toxicity were observed during the study period. On day 2 (1h after the second application), the animals treated with a test item concentration of 2% showed an erythema of the ear skin (score 1). Animals treated with 0.5 and 1% test item concentration did not show any signs of local skin irritation.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant decrease in ear weights was observed in the mid and the high dose groups in comparison to the vehicle control group (p<0.05). Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups reached or exceeded this threshold.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test item 5-Methyl-3-vinyloxazolidin-2-on was not a skin sensitiser under the test conditions of this study.

Executive summary:

In this study the test item 5-Methyl-3-vinyloxazolidin-2-on was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solutions at different concentrations were prepared in the vehicle PG (propylene glycol).

The local lymph node assay is recommended by international test guidelines (e.g., OECD) as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations.

For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 1, and 2% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by three pre-experiments).

The animals showed neither signs of systemic toxicity nor mortality during the course of the study. On day 2 (1h after the second application), the animals treated with a test item concentration of 2% showed an erythema of the ear skin (score 1). Animals treated with 0.5 and 1% test item concentration did not show any signs of local skin irritation. A statistically significant decrease in ear weights was observed in the mid and the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups reached or exceeded this threshold.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 2.09, 2.77, and 2.40 were determined with the test item at concentrations of 0.5, 1, and 2% (w/w) in PG, respectively. A clear dose response was not observed. No statistical outlier was detected in both outlier tests. However, the DPM value determined for animal No. 1 (205.5 DPM) was outside the range of the historical vehicle controls (for PG: mean: 870.6 dpm, min. 305.5 DPM, max. 1661.5 DPM, standard deviation: 308.9 DPM). However, this value was not excluded from calculations, since exclusion of this value had no biological relevance and would not have changed the overall study result.

Although a statistically significant increase in the mid dose group of the DPM value and also in the low and high dose group of the lymph node cell count was observed in comparison to the vehicle control group, this was not considered to be biologically relevant as the S.I. determined for these concentrations did not exceed the threshold value of 3. A statistically significant or biologically relevant increase in lymph node weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any dose group.

The test item 5-Methyl-3-vinyloxazolidin-2-on was thus not a skin sensitizer under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitizing potential of 5 -methyl-3 -vinyloxazolidin-2 -one was investigated in a local lymph node assay according to OECD guideline 429 (BASF SE, 2015). The test was performed using test item concentrations of 0.5, 1, and 2% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by three pre-experiments).

The animals showed neither signs of systemic toxicity nor mortality during the course of the study. On day 2 (1h after the second application), the animals treated with a test item concentration of 2% showed an erythema of the ear skin (score 1). Animals treated with 0.5 and 1% test item concentration did not show any signs of local skin irritation. A statistically significant decrease in ear weights was observed in the mid and the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups reached or exceeded this threshold. In this study Stimulation Indices (S.I.) of 2.09, 2.77, and 2.40 were determined with the test item at concentrations of 0.5, 1, and 2% (w/w) in. A clear dose response was not observed. Although a statistically significant increase in the mid dose group of the DPM value and also in the low and high dose group of the lymph node cell count was observed in comparison to the vehicle control group, this was not considered to be biologically relevant as the S.I. determined for these concentrations did not exceed the threshold value of 3. A statistically significant or biologically relevant increase in lymph node weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any dose group. The test item 5 -methyl-3-vinyloxazolidin-2-one was thus not a skin sensitizer under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the available data, the classification criteria according to Regulation (EC) No 1272/2008 (CLP) are not met.