Sebacohydrazide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 June 2018 - 27 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

09 October 2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sebacohydrazide

EC Number:

213-126-8

EC Name:

Sebacohydrazide

Cas Number:

925-83-7

Molecular formula:

C10H22N4O2

IUPAC Name:

sebacohydrazide

Test material form:

solid: particulate/powder

Details on test material:

Test item name (as stated in report): SDH
Test item storage: at room temperature protected from light

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE
- Bovine eyes from young cattle were obtained from the slaughterhouse
- Eyes were collected and transported in physiological saline in a suitable container under cooled conditions

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 307.4 to 376.9 mg per cornea

Duration of treatment / exposure:

240 ± 10 minutes at 32 ± 1°C

Duration of post- treatment incubation (in vitro):

Incubation with sodium fluoresceine 90 ± 5 minutes at 32 ± 1°C for permeability determination

Number of animals or in vitro replicates:

3 replicates for the negative, positive, and treatment group

Details on study design:

TEST SITE
- Isolated bovine cornea

TREATMENT METHOD: The medium from the anterior compartment was removed and 750 μL of the negative and the positive control. SDH was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (307.4 to 376.9 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ±10 minutes at 32 ± 1°C

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM)

SCORING SYSTEM:
- Opacity was measured by the diminution of light passing through the cornea, measured as illuminance by a light meter
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

ACCEPTABILITY OF THE RESULTS
The test is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

First experiment

Value:

4.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test was inconclusive as results were spread over 2 categories (17, -3.2, -0.4, respectively)

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Second experiment

Value:

-1.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Two experiments were performed
- First experiment:
In vitro irritancy scores: -3.2, -0.4 and 17. The test was inconclusive and a repeat experiment was performed
Opacity values: -3.8 to 9.1
Permeability values: 0.002 to 0.498.

- Second experiment:
In vitro irritancy scores: -2.8 to -0.1
Opacity values: -3.3 to -0.6
Permeability values: 0.031 to 0.044

The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 157 and 153 in the first and second experiment, respectively and within two standard deviations of the current historical positive control mean
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range in both experiments

Any other information on results incl. tables

- The corneas were translucent after 240 minutes of treatment with SDH in both experiments

- No pH effect of the test item was observed on the rinsing medium

Applicant's summary and conclusion

Interpretation of results:

other: Not irritating

Remarks:

according to EC No 1272/2008

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline and GLP principles, it is concluded that SDH is not irritant or corrosive for the eye

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed according to OECD guideline 437 and in accordance with GLP principles. Two experiments were performed. SDH was applied undiluted (307.4 to 376.9 mg) for 240 minutes. In the first experiment, the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range and the mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 157 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The mean in vitro irritancy score was 4.3 after 40 hours of treatment with the test item. Since the results were spread over 2 categories (17, -3.2, -0.4, respectively), the test was inconclusive and a repeat experiment was performed.

In the second experiment, the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range and the mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 153 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

SDH did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.8 after 4 hours of treatment.

In conclusion, since SDH induced an IVIS ≤ 3 in 5 out of 6 cornea’s, no classification is required for eye irritation or serious eye damage.