Zinc isodecyl phosphorodithioate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted July 26 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc isodecyl phosphorodithioate

EC Number:

246-618-6

EC Name:

Zinc isodecyl phosphorodithioate

Cas Number:

25103-54-2

Molecular formula:

C20H43O2PS2.1/2Zn

IUPAC Name:

zinc bis[O,O-bis(8-methylnonyl) dithiophosphate]

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Vitelco’s, Hertogenbosch, The Netherlands)
- Number of animals: Not specified.
- Characteristics of donor animals (e.g. age, sex, weight): Young cattle.
- 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.
- Time interval prior to initiating testing: 1 hour
- indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects by removing them from the physiological saline and holding them up to the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: Not applicable.

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): 750 µL was introduced onto the epithelium of the cornea
- Concentration (if solution): Test material was tested neat.

Duration of treatment / exposure:

The test consisted of topical application of the test item on the epithelium of the bovine cornea for 10 minutes. After exposure the cornea was thoroughly rinsed to remove the test item and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

Duration of post- treatment incubation (in vitro):

90 +- 5 minutes at 32 +-1°C

Number of animals or in vitro replicates:

Three test replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Preparation: The eyes were checked for defects such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. The isolated corneas were then stored in a petri dish with Earle’s Minimum Essential Medium (cMEM) containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder. The anterior half of the holder was positioned on top of the cornea and tighten with screws. The compartments of the corneal holder were filled with cMEM at 32 +/- 1 °C.
Selection: The corneas were incubated for a minimum of 1 hour at 32 +/-1 °C. The opacity of each cornea was read against a cMEM 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were checked for defects such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Physiological saline

SOLVENT CONTROL USED (if applicable): Not applicable

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 μl of test item was applied to the epithelium of the bovine cornea for 10 minutes.

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: 90 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Not specified

POST-EXPOSURE INCUBATION: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.
The light was measured as illuminance (I = luminous flux per area, unit: lux) by a Opacitometer.

- Corneal permeability: Microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Not specified.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
The decision criteria for the IVIS cut-off values for identifying test chemicals as inducing serious eye damage was used for the study.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

2.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

-0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

other: Permeability

Run / experiment:

Mean

Value:

0.03

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

A summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score
Negative control	-0.1	-0.005	-0.2
Positive control (Ethanol)	27	0.802	39
Test item	0.5	0.030	1.0

Historical control data for the BCOP Studies

Negative Control				Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9-3.0	-0.016-0.042	-2.8-3.0	34.7-78.2
Mean	0.08	0.01	0.17	56.01
SD	1.04	0.01	1.14	12.51
n	84	77	78	55

SD= Standard deviation

N= Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of August 2014 to August 2017.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not an eye irritant under the conditions of the bovine corneal opacity and permeability test guideline.

Executive summary:

The study is assigned a reliability score of 1 (reliable without restrictions) as it followed OECD Guideline 437: Bovine Corneal Opacity and Permeability Test Method and is also compliant with GLP. 750 µL of the undiluted test item was applied directly on top of the corneas for 10 minutes.

The positive control mean in vitro irritancy score (39) was within two standard deviations of the current historical positive control mean. The negative control values for opacity and permeability were less than the upper limits of the laboratory historical range. The validity criteria for the study were therefore met.

The test item induced an In vitro irritancy score of less than 3, which concludes no classification is required for eye irritation or serious eye damage.