5-methyl-1,3-oxazolidin-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation, other

Remarks:

LLNA

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

5-methyl-1,3-oxazolidin-2-one

EC Number:

810-292-9

Cas Number:

1072-70-4

Molecular formula:

C4H7NO2

IUPAC Name:

5-methyl-1,3-oxazolidin-2-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 5-methyl oxazolidin-2-one
- Physical state: liquid, colorless clear
- Analytical purity: 98.0 area %
- Lot/batch No.: EWALD-00441

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Horst / The Netherlands
- Age at study initiation: Pre-test and Main study: 9 - 10 weeks
- Housing: group ( Makrolon Type II (pre-test) / III (main study), with wire mesh top)
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): approx. 45-65%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

25, 50, 100 %

No. of animals per dose:

5

Details on study design:

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25% and 50% (w/w) in PG, as well as undiluted (100%). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 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. 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.

Positive control substance(s):

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

Statistics:

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 DecisionTree_2.Rnw was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers (performed with validated program R Script Outlier.Rnw).

Results and discussion

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.

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: In this study Stimulation Indices (S.I.) of 1.01, 1.98 and 2.63 were determined with the test item at concentrations of 25% and 50% (w/w) in PG, as well as with the undiluted test item (100%) respectively. A clear dose response was observed.

Any other information on results incl. tables

Lymph Node Weights and Cell Counts

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant, but not biologically relevant increase in lymph node–cell counts was observed in the high dose group in comparison to the vehicle control 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. The index determined for the lymph node cell count exceeded this threshold in the high dose group (index of 1.79). For the lymph node weights, no statistically significant increase was observed in any dose group in comparison to the vehicle control group.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached of exceeded in any of the treated groups.

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (DMF)	540.3	165.9	1.00
25% 5-methyl oxazolidin-2-one	545.7	270.6	1.01
50% 5-methyl oxazolidin-2-one	1068.3	210.3	1.98
100% 5-methyl oxazolidin-2-one	14.23.5	580.3	2.63*

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, except for test group1 for which 4 animals were included)

*Mean DPM value for the group was according to the ANOVA (Dunnett-test) significantly higher than the corresponding control value. The p value for the analysis was <0.05

Applicant's summary and conclusion

Conclusions:

The test item 5-methyl oxazolidin-2-one was not a skin sensitiser under the test conditions of this study.