Methyl o-toluate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-05-03 till 2015-07-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Test material

Test material information

Constituent 1

Reference substance name:

Methyl o-toluate

EC Number:

201-932-2

EC Name:

Methyl o-toluate

Cas Number:

89-71-4

Molecular formula:

C9H10O2

IUPAC Name:

methyl o-toluate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Ylanganate
- Storage condition of test material: ambient temperature (15-25ºC)
- Physical state: Colorless liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other:

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm™ (EPI-200) skin model consisted of normal human epidermal keratinocytes from one single donor, derived from neonatal-foreskin tissue. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2). The skin models are commercially available and were obtained from MatTek In Vitro Life Science Laboratories (IVLSL), Slovakia.

Amount/concentration applied:

50 ul

Duration of treatment / exposure:

Skin corrosion test: 3 min and 60 min.
Skin irritation test: 60 min and 42h of post exposure

Duration of post-treatment incubation (if applicable):

Skin corrosion test: Immediately after exposure the viability of the epidermal cells was assessed using the MTT test.
Skin irritation test: Viability of the epidermal cells was assessed using the MTT test after 42h of post exposure

Number of replicates:

2

Test system

Controls:

other: Positive controls: Skin corrosion test 8 M potassium hydroxide solution and skin irritation test: 5% aqueous sodium dodecyl sulphate (SDS). Negative controls: Skin corrosion test: Milli-Q water and skin irritation test: phosphate buffered saline (PBS).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion: 3 minutes exposure

Value:

97

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion: 60 minutes exposure

Value:

16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Irritation at 60 minutes exposure and 42h post exposure

Value:

5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

In vivo

Results

Results 1

Irritation parameter:

other: Tissue viability (% of negative control)

Basis:

mean

Time point:

other: 3 min

Score:

97

Max. score:

100

Remarks on result:

other: Skin corrosion test (SD = 13)

Results 2

Irritation parameter:

other: Tissue viability (% of negative control)

Basis:

mean

Time point:

other: 60 min

Score:

16

Max. score:

100

Remarks on result:

other: Skin corrosion test (SD = 4)

Results 3

Irritation parameter:

other: Tissue viability (% of negative control)

Basis:

mean

Time point:

other: 60 min

Score:

5

Max. score:

100

Remarks on result:

other: Skin irritation test (SD = 0)

Irritant / corrosive response data:

Preliminary tests
- At the end of the incubation period of the test substance with a MTT solution, the MTT solution had neither turned blue/purple nor showed a blue/purple precipitate, indicating that the test substance did not have the potential to reduce MTT. Therefore, no additional controls were required in the in vitro skin irritation test. During the mesh compatibility test it was concluded that the test substance did not have the potential to damage the nylon mesh and therefore the nylon mesh was used in the in vitro skin corrosion test to facilitate equal distribution of the test substance over the skin membrane surface. The preliminary test to determine if the test substance has the potential to stain the skin membranes during the exposure (as required according OECD guideline no. 431) was not performed, because no tissue staining was observed during the in vitro skin irritation test.

In vitro skin corrosion test
- The mean OD of the negative control (MilliQ) and the positive control (8 M KOH) demonstrated the expected response. The CV of the test groups that showed mean viability between 20% and 100% was ≤ 30%. All acceptance criteria were met and therefore the test was considered valid.
- At the end of the 3 min and 60 exposure, the skin membranes of the positive controls were detached from the insert, most likely as a result of the corrosiveness of the positive control.
- One skin membrane of the negative control of the 3 min exposure group (i.e. replicate NC3-2) was excluded from calculations due a technical error. Prior to OD measurement at the end of the extraction with MTT extractant, it was observed that the formazan was inefficiently extracted from the skin membrane (i.e. the skin membrane was still purple stained, whereas skin membranes from other groups were not stained because the formazan was efficiently extracted from the skin membranes). The purple staining of the membrane of replicate NC3-2 confirmed viability of the skin membrane and the reason for the inefficient extraction is unknown.

In vitro skin irritation test
- The mean OD of the blank, the negative control (PBS) and the positive control (5% SDS) demonstrated the expected response. The SD calculated from individual tissue viability percentages of the three replicates was <18%. All acceptance criteria were met and therefore the test was considered valid.

Applicant's summary and conclusion

Interpretation of results:

other: A skin irritant Category 2

Remarks:

according to the CLP Regulation EC 1272/2008 and its updates

Conclusions:

Under the test conditions (OECD 431, 439 and GLP) the test substance is considered to be a skin irritant.

Executive summary:

In accordance to OECD guideline 431, 439 and GLP the test substance was examined for its in vitro skin corrosion and irritation potential using EpiDerm™ reconstructed skin membranes. In the in vitro skin corrosion test, the skin membranes were topically exposed to the undiluted test substance for 3 min and 60 min. Immediately after exposure the viability of the epidermal cells was assessed using the MTT test. In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for 60 min. Viability of the epidermal cells was assessed using the MTT test after 42 h of culture. Per test group, tests were performed in duplicate. In both tests negative and positive controls were run in parallel. In the skin corrosion test the mean viability of the skin membranes was 97 ± 13% after 3 minutes and 16 ± 4% after 60 minutes. In the skin irritation test the mean viability was 5 ± 0% compared to the concurrent negative control group. Based on the results obtained in the present study the test substance is considered to be a skin irritant.