ACETIC ACID, 2-(3-CHLORO-5-CYANOPHENOXY)-, ETHYL ESTER

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9th September 2013 to 12th November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Principles of method if other than guideline:

The MalTek EpiDerm™ MTT Viability Assay has been demonstrated to be a quantitative method for assessing potential skin hazards.

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

ethyl 2-(3-chloro-5-cyanophenoxy)acetate

EC Number:

815-596-5

Cas Number:

1613307-27-9

Molecular formula:

C11H11CINO3

IUPAC Name:

ethyl 2-(3-chloro-5-cyanophenoxy)acetate

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: not specified

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test articles were dosed neat.
A Negative Control, 100 μI of tissue culture water, was tested concurrently.
A Positive Control (1% Triton" X-100) was tested concurrently.

Duration of treatment / exposure:

The test article, the positive control (1% Triton" X-100), and the negative control tissue culture water), were treated in duplicate EpiDermTM tissues for 1,4 and 24 hours. A Negative Control (undosed tissues) was tested at 4 hours. A Positive Control (1% Triton" X-100) was tested at 4 and 9 hours.

Duration of post-treatment incubation (if applicable):

At the end of the selected exposure periods, each EpiDerm™ tissue was rinsed with phosphate
buffered saline (PBS) and transferred to a 24-well plate containing 300 μI of MTT solution (1 mg/ml MTT in DMEM). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well.

Number of replicates:

Duplicate

Test system

Details on study design:

Test Article Reduction of MTT:
For each test article, 100 mg of the test article were mixed with 1 ml of MTT solution (1 mg/ml Methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium (DMEM»). A Negative Control, 100 μI of tissue culture water, was tested concurrently. The solutions were incubated at room temperature in the dark for 60 minutes. After incubation, the solutions were visually inspected for purple coloration, which indicates that the test article reduced MTT. Since tissue viability is based on MTT reduction, direct reduction by a test article can exaggerate viability, making a test article seem less irritating than it really is None of the test articles were found to have reduced MTT and the assay continued as per the protocol.

Tissue Viability (MTT Reduction):
At the end of the selected exposure periods, each EpiDerm™ tissue was rinsed with phosphate
buffered saline (PBS) and transferred to a 24-well plate containing 300 ~I of MTT solution (1 mg/ml MTT in DMEM). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. An aliquot of the extracted MTT formazan was measured at 540 nrn using a plate reader, subtracting the absorbance at a reference wavelength of 690 nm.

Analysis of Data:
The mean absorbance value for each time point was calculated from the optical density (00) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability = 100 X (00 sample/OD Negative Control)

The ET50, the time at which the EpiDerm ™ tissue viability was reduced 50% compared to Control tissues, was then determined using a macro in Microsoft Excel 5.0, provided by MatTek, using the equation: V=a+blogt
Where V =percentage viability, t =time in hours, and a and b are constants that can be determined by using the viability data for two different exposure times of the test article to the tissue. These exposure times must yield viabilities that flank 50%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: ET50

Run / experiment:

L-005183690-000F003

Value:

21

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Very mild

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

L-005183690-000F003 - Very Mild

Executive summary:

OBJECTIVE: To provide an estimate of dermal irritation potential using an alternative to the Draize methodology. The MalTek EpiDerm™ MTT Viability Assay has been demonstrated to be a quantitative method for assessing potential skin hazards.

METHOD SYNOPSIS: MatTek EpiDerm ™ tissue samples were treated in duplicate with the test articles and Positive Control for various exposure times listed below. Negative Controls (undosed tissues) were tested at4 hours only. Following treatment, the viability of the tissues was determined using Methyl thiazole tetrazolium (MTT) uptake and reduction. The absorbance of each sample was measured at 540 nm using a reference wavelength of 690 nm. The viability was then expressed as a percent of Control values. The mean percent viability for each time point was used to calculate an ET50, which represents the time at which the EpiDerm TM tissue viability was reduced 50% compared to Negative Control tissues. The ET50 scores were converted to an irritancy classification.

SUMMARY/CONCLUSION:L-005183690-000F003  Very Mild