Methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 - 29 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted by GLP accredited laboratory. Method according to OECD guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: Human Skin Model Test (adopted 13 April 2004).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

bis: "in vitro Skin Corrosion: Human Skin Model Test" - Regulation (EC) no. 440/2008.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

EC Number:

700-527-2

Cas Number:

1271488-66-4

Molecular formula:

C12H20O3

IUPAC Name:

methyl trans-3-oxo-2-pentylcyclopentanecarboxylate

Test animals

Species:

human

Details on test animals or test system and environmental conditions:

The incubations took place in the dark and in air with a humidity of 56-86% containing 0.5±0.5% carbon dioxide at 36.8-37.3°C.

Test system

Type of coverage:

open

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Details on study design:

Prior to the analysis, the test substance was checked for possible direct reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1) (MTT). 99.5 µl of the test substance was added to 1 ml MTT and incubated for 1h at room temperature. A negative control of sterile Milli-Q water was included.
 
The skin tissues were placed into a 6-well plate on 0.9ml Dulbecco's Modified Eagle's Medium (DMEM) per well and incubated for 105 minutes at 37°C in air. The medium was replaced with fresh DMEM just before the test substance was applied.
 
4 samples plus a negative control of Milli-Q water and positive control of 8N potassium hydroxide was included in the test. Two tissue samples were exposed for 3 minutes and two samples for 1-hour to 50 µl test substance, that was added on top of the tissue. After the exposure, the tissues were rinsed with phosphate buffered saline
 
The DMEM medium was replaced by 300 µl MTT medium and the tissues were incubated for 3 hours under the same environmental conditions. The tissues were subsequently washed with PBS and the formed formazan was extracted with 2 ml isopropanol overnight at room temperature. The amount of Formazan was determined spectrophotometrically at 540 nm in triplicate. The cell viability was measured against the mean of the negative control.

Results and discussion

In vivo

Irritant / corrosive response data:

The test substance was visually checked for possible direct MTT reduction causing a colour change. No interaction with MTT was observed.
Details of the data and the mean of the spectroscopic measurements can be found in Table 1. Table 2 presents the mean relative tissue viability being the mean of the absorption of the test substance divided by the negative control mean for each application. Since the mean relative tissue viability of the test substance was not below 50% after an exposure duration of 3-minutes and 15% after 1 hour, the test substance is not considered corrosive.

Any other information on results incl. tables

Table 1  Absorptions (OD540) in the in vitro skin corrosion test
	3-minute application					1-hour application
	A	B	Mean	SD		A	B	Mean	SD
Negative control	0.960	0.998	0.972	0.033		1.040	1.076	1.044	0.035
	0.953	1.009				1.008	1.063
	0.934	0.979				1.011	1.067
Test substance	1.008	1.063	1.031	0.050		1.101	1.098	1.099	0.001
	0.999	1.076				1.102	1.103
	0.981	1.060				1.097	1.094
Positive control	0.084	0.075	0.077	0.002		0.055	0.072	0.580	0.004
	0.071	0.078				0.057	0.056
	0.072	0.081				0.051	0.054
Duplicate exposures are indicated by A and B

Table 2 Mean tissue viability as % of negative control in the in vitro skin corrosion

Application	3-minute		1-hour
	%		%
Negative control	100		100
Test substance	106		105
Positive control	8		6

Table 3 Historical control data for the in vitro skin corrosion studies (Data collected from July 2007 to August 2010)

	Negative control			Positive control
	3-minute	1-hour		3-minute	1-hour
Range	1.226-2.003	1.365-2.007		0.075-.278	0.057-.242
mean	1.60	1.60		0.13	0.11
SD	0.15	0.12		0.05	0.04
n	58	58		58	58

SD: standard deviation; n: number of observations

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: OECD GHS

Conclusions:

The test is valid and the test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions used.

Executive summary:

An in vitro skin corrosion test was conducted on a human skin model according to the OECD guidelines. The basis of the test is that the cell viability was determined by using the MTT reduction assay. The test substance was applied undiluted on top of the skin tissue for 3 minutes and 1 hour. After the exposure, the tissue was incubated with MTT and the formazan that was produced from MTT was spectroscopically determined at 540 nm. The relative mean tissue viability of the test substance expressed as the absorption of the test substance against the negative control showed that the test substance has the same tissue viability as the negative control. The test substance is therefore not considered as corrosive.