Propan-2-yl-oxocyclobutane carboxylate

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12-12-2018 to 22-01-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity: Fixed Dose Procedure)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Test material

Test material information

1

Reference substance name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

EC Number:

834-970-9

Cas Number:

130111-95-4

Molecular formula:

C8H12O3

IUPAC Name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

Test material form:

liquid

Details on test material:

Batch (Lot) Number: GR13224
Expiry date: 31 January 2020 (expiry date)
Physical Description: Colourless to pale yellow liquid
Purity/Composition: 99.6%
Storage Conditions: At room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: Crl: WI(Han)
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany
Number of Animals: 3 females (nulliparous and non-pregnant).
Age at the Initiation of Dosing: Young adult animals (approximately 10 weeks old) were
selected.
Weight at the Initiation of Dosing: 177 to 196 g.

Justification for Test System and Number of Animals
The Wistar Han rat was chosen as the animal model for this study as recognized by
international guidelines as a recommended test system. The test method and number of
animals were based on the test guidelines.
The study plan was reviewed and agreed by the Animal Welfare Body of Charles River
Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license
AVD2360020172866 approved by the Central Authority for Scientific Procedures on

Animal Identification
At study assignment, each animal was identified using a tail mark with indelible ink.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at
least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician
according to body weights, with all animals within ± 20% of the sex mean. Animals in poor
health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed and any assigned animal
considered unsuitable for use in the study were replaced by alternate animals obtained from
the same shipment and maintained under the same environmental conditions.
The disposition of all animals was documented in the study records.

Husbandry
Housing
On arrival, animals were group housed (up to 5 animals of the same sex together) in
polycarbonate cages (Makrolon MIV type; height 18 cm.) and following assignment to the
study, animals were individually housed in polycarbonate cages (Makrolon MIII type; height
18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS -
J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
The room in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly
labeled.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were
maintained. The actual daily mean temperature during the study period was 20 to 21°C with
an actual daily mean relative humidity of 27 to 53% (see deviations in Appendix 3). A
12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with
100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was
provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental
contaminants. Results of the analysis were provided by the supplier and are on file at the Test
Facility.
It is considered that there were no known contaminants in the feed that would interfere with
the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the
Test Facility.
It is considered that there were no known contaminants in the water that would interfere with
the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri,
Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted
by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations
or treatments were required.

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

A single dose of test item was administered to the appropriate animals by dermal application
on Day 1. One day before dosing, an area of approximately 5x7 cm on the back of the
animals was clipped. The test item was applied in an area of approximately 10% of the total
body surface, i.e. approximately 18 cm2 for females. The test item was held in contact with
the skin with a dressing, consisting of a surgical gauze patch (Surgy 1D), successively
covered with Coban elastic bandage. A piece of Micropore tape was additionally used for
fixation of the bandages in females only. The application period was 24 hours, after which
the dressing was removed and the skin cleaned of residual test item using water. For one
animal (no. 2), it was noted the day after application that the dressings had fallen of the
animal. Actual exposure time for this animal was between 6 hours and 17 minutes and 15
hours and 8 minutes. As no significant difference was noted between results of the animals,
the shorter exposure time was concluded not to have affected the outcome of the study.
The dose volume for each animal was based on the body weight measurement prior to dosing.
Dose volume (mL/kg body weight) was calculated as follows:
Dose level (g/kg) / spec.gravity or density (g/mL).
The dosing formulations were stirred continuously during dose administration.

Justification of Route and Dose Levels
The dermal route was selected as it is a possible route of human exposure during
manufacture, handling or use of the test item. The dose levels were based on the OECD test
guidelines.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3 per sex per dose

Control animals:

not required

Details on study design:

Range Finding Study
A range finding study was performed in order to select the dose causing no mortality or
significant toxicity to be used in the main study. One animal was dosed at 2000 mg/kg.

Main Study
Based on the results of the range finding study, two animals were dosed at 2000 mg/kg.

In-life Procedures, Observations, and Measurements
Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity
twice daily, in the morning and at the end of the working day. Animals were not removed
from cage during observation, unless necessary for identification or confirmation of possible
findings.

Clinical Observations
Postdose Observations
Postdose observations were performed at periodic intervals on the day of dosing (at least three
times) and once daily thereafter, except for the animal in the range finder for which signs
were not recorded on Day 15, see deviation in Appendix 2. The observation period was 14
days.
All the animals were examined for reaction to dosing. The onset, intensity and duration of
these signs was recorded (if appropriate), particular attention being paid to the animals during
and for the first hour after dosing.

Body Weights
Animals were weighed individually on Day 1 (predose), 8 and 15.

Irritation
The skin reactions were assessed approximately 24, 48 and 72 hours after the removal of the
dressing and test item. Adjacent areas of untreated skin of each animal served as controls.
The following numerical scoring system was used in the scoring of skin reactions:
Erythema and eschar formation:
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) * 4
*. Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for
erythema (= 4) is given.
Oedema formation:
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimeter) 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) 4

Terminal Procedures
All animals were sacrificed by oxygen/carbon dioxide procedure at the end of the observation
period. All animals assigned to the study were subjected to necropsy and descriptions of all
internal macroscopic abnormalities were recorded.

Statistics:

All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.
The dermal LD50 value of the test item was ranked within the following ranges: 0-50, 50-
200, 200-1000 or 1000-2000 mg/kg b.w. or as exceeding 2000 mg/kg b.w.
The results can be evaluated according to the Globally Harmonized System of Classification
and Labelling of Chemicals (GHS) of the United Nations (including all amendments) and the
Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16
December 2008 on classification, labelling and packaging of items and mixtures (including
all amendments).

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred.

Clinical signs:

other: No clinical signs were noted for any of the animals at any time point.

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals.

Other findings:

No irritation was noted for any of the animals at any time point.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The dermal LD50 value of PF-06238566 in Wistar Han rats was established to exceed 2000
mg/kg body weight
Based on these results, PF-06238566 does not have to be classified and has no obligatory
labelling requirement for acute dermal toxicity according to the Globally Harmonized System
of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including
all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and
packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this study was to determine the potential toxicity of  PF-06238566, when

given by a single dermal dose.

The study was carried out based on the guideline described in:

- OECD No. 402 (2017) "Acute Dermal Toxicity".

Initially,  PF-06238566 was administered to a single female Wistar Han rat by a single dermal

application at 2000 mg/kg body weight for 24 hours in a range finder study. Based on the

results, the main study was performed by dosing two female Wistar Han rats at 2000 mg/kg.

All animals were subjected to daily observations and weekly determination of body weight.

Macroscopic examination was performed after terminal sacrifice (Day 15).

No mortality occurred.

No clinical signs were noted for any of the animals at any time point.

No irritation was noted for any of the animals at any time point.

The mean body weight gain during the observation period was within the range expected for

rats used in this type of study.

No abnormalities were found at macroscopic post  mortem  examination of the animals.

The dermal LD50 value of  PF-06238566 in Wistar Han rats was established to exceed 2000

mg/kg body weight

Based on these results,  PF-06238566 does not have to be classified and has no obligatory

labelling requirement for acute dermal toxicity according to the Globally Harmonized System

of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including

all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and

packaging of items and mixtures (including all amendments).