3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 20 February 2019 to 20 March 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The relative humidity in the animal room was for a few hours between approximately 13 - 45 % instead of 45 - 65% as stated in the study plan. This deviation to the study plan is not considered to affect the validity of the study.

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:

-

EC Number:

482-280-8

EC Name:

-

Cas Number:

449177-94-0

Molecular formula:

C8H11F5O2

IUPAC Name:

3-methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane

Test material form:

liquid

Details on test material:

Lot No: 2231AM-68E
Expiry date: 01 January 2019
Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V, Inc., Postbus 6174, 5960 AD Horst, The Netherlands.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Pre-test: 11 to 12 weeks; Main Test: 9 to 10 weeks
- Weight at study initiation: Not Stated
- Housing: Caged by group in cages with granulated soft wood bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2°C
- Humidity (%): 45 to 65%
- Photoperiod (hrs dark / hrs light): 12 hours in light, 12 hours in darkness

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Control = vehicle only
Low dose = 25% v/v
Mid dose = 50% v/v
High dose = 100% v/v

No. of animals per dose:

Four per dose (Plus four for the control)

Details on study design:

PRELIMINARY TEST

The systemic toxicity and irritancy potential of the test item was assessed in a preliminary screening test performed using two mice at 50 or 100% v/v test substance respectively. No signs of systemic toxicity were observed; however, very slight erythema of the ear skin was observed in both animals. Additionally, the animal treated with 100% test item concentration showed scaly ears on day 6. No signs of excessive local skin erythema were present in both animals.

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

MAIN TEST

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25% v/v and 50% v/v, in acetone/olive oil (4+1 v/v), and 100% (undiluted). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (approximately 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).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.98 µCi of 3H-methyl thymidine (equivalent to 79.9 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

Preparation of Single Cell Suspensions
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). 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.

Determination of cellular proliferation (incorporation of 3HTdR)
The precipitates were 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 β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OBSERVATIONS

Clinical Observations
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

Determination of Ear Thickness
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometre.

Determination of ear weights
In the pre-test, after the lymph nodes have been excised, both ears 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 per animal using an analytical balance. The values obtained were taken down manually.

Determination of Body Weights
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment).

Positive control substance(s):

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

Results and discussion

Positive control results:

The postive control (run as a separate study) responded as expected and confirmed the responsiveness of the test system in the laboratory.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.93

Test group / Remarks:

25% v/v test substance

Results 2

Key result

Parameter:

SI

Value:

0.76

Test group / Remarks:

50% v/v test substance

Results 3

Key result

Parameter:

SI

Value:

0.66

Test group / Remarks:

100% v/v test substance

Any other information on results incl. tables

There were no deaths on the study.  No signs of systemic toxicity were observed during the study period. On day 3, animals treated at all test item concentrations showed a very slight erythema of the ear skin.

Body weight change of the test 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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was not a skin sensitiser under the test conditions of this study.

Executive summary:

Introduction

The study was performed to assess the skin sensitisation potential of the test substance in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following guidelines:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)
  
  
• Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008 (updated 06 July 2012)

 

Method

Following preliminary screening test animals showed a very slight erythema of the ear skin. No clinical signs of toxicity were noted at a concentration of 100% and 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 μL (25 μL per ear) of the test item at 25% and 50%, in acetone/olive oil (4 +1 v/v), and 100% (undiluted). A further group of four animals was treated with acetone/olive oil 4:1 alone.

Results

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3, animals treated at all test item concentrations showed a very slight erythema of the ear skin.

The Stimulation Indices expressed as the mean radioactive incoporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v)	Stimulation Index	Result
25	0.93	Negative
50	0.76	Negative
100	0.66	Negative

 

 

Conclusion

The test item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was not a skin sensitiser under the test conditions of this study.