Carbamic acid, N-[(butylthio)thioxomethyl]-, butyl ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

(2009)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

(2010)

Deviations:

no

Principles of method if other than guideline:

- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

EC Number:

691-061-8

Cas Number:

1001320-38-2

Molecular formula:

C10H19NO2S2

IUPAC Name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

Test material form:

solid

Remarks:

yellow grease-like solid

Specific details on test material used for the study:

Molecular formula: C10H19NO2S2
Name: n-butoxycarbonyl n-butyl dithiocarbamate (active ingredient)
CAS Number: 1001320-38-2

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 750 µl per cornea

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 10% (w/v) Benzalkonium Chloride

Duration of treatment / exposure:

10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The isolated corneas were stored at 32 ± 1°C in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group

NUMBER OF REPLICATES - 3

NEGATIVE CONTROL USED - Physiological saline

POSITIVE CONTROL USED - 10% (w/v) Benzalkonium Chloride

APPLICATION DOSE AND EXPOSURE TIME - 750 µl for 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: washed once with Hanks’ balanced salt solution and thereafter with cMEM
- POST-EXPOSURE INCUBATION: 120 min

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

1

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean in vitro irritancy score of the negative control was 0 and of the positive control (10% (w/v) Benzalkonium Chloride) was 129.
The corneas treated with S-10903 showed opacity values ranging from 0 to 3 and permeability values ranging from -0.002 to 0.003. The corneas were clear after the 10 minutes of treatment with S-10903. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.1 to 3.0 after 10 minutes of treatment with S-10903.

Any other information on results incl. tables

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 129 and within the historical positive control data range. Negative and positive controls were valid and it was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test performed according to OECD 437 guideline and GLP principles.

Executive summary:

The substance was screened for the eye irritancy potential using the BCOP test performed according to OECD 437 guideline and GLP principles.

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 129 and within the historical positive control data range. Negative and positive controls were valid and it was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The substance S-10903 did not induce ocular irritation through both endpoints, opacity and permeability, resulting in a mean in vitro irritancy score of 1.0 after 10 minutes of treatment.

Based on the results it is concluded that S-10903 is not a severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test.

Acceptability test and interpretation results:

The BCOP test method is recommended by authorities to identify the eye hazard potential of a test chemical. This test method is also recommended to identify chemicals that do not require classification for eye irritation or serious eye damage, as defined by the UN GHS, in case IVIS ≤ 3.

As in the present study with S-10903 an IVIS of 1.0 was obtained, indicating no irritating properties for S-10903, further testing for eye irritating properties is considered redundant and ethically not justified.