Isophthalohydrazide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2017 - 24 October 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:

other: OECD guideline 437 “Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants”

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Isophthalohydrazide

EC Number:

220-425-7

EC Name:

Isophthalohydrazide

Cas Number:

2760-98-7

Molecular formula:

C8H10N4O2

IUPAC Name:

isophthalohydrazide

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White crystal powder
Test item storage: At room temperature

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: approx. 327.4 -361.9 mg per cornea

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

POSITIVE CONTROL
Amount applied: 750 µl per cornea
Concentration: 20% (w/v) Imidazole

Duration of treatment / exposure:

240 ± 10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM)
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed by incubation in cMEM. Then, corneas were incubated in sodium-fluorescein solution for 90 ± 5 minutes (both incubations at 32 ± 1°C).

- 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

DATA EVALUATION:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: catgeory 1);
For a test substance that induces an IVIS >3 and ≤ 55, no prediction on irritant potency can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Single run

Value:

-1.7

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:

The individual in vitro irritancy scores for the negative controls ranged from 1.3 to 3.7. The individual positive control in vitro irritancy scores ranged from 97 to 158. The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas treated with IDH showed opacity values ranging from -3.0 to -1.6 and permeability values ranging from 0.013 to 0.063. The corneas clear after the 240 minutes of
treatment with IDH. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.8 to -0.8 after 240 minutes of treatment with IDH.

See table for mean scores

Any other information on results incl. tables

Table: Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^[1]^,[2]
Negative control	2.1	0.014	2.4
Positive control	95	2.802	137
IDH	-2.3	0.042	-1.7

[1]Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

[2]In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

            

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, it is concluded that IDH is not irritant or corrosive for the eye and does not need to be classified.

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with IDH according to OECD guideline 437 and GLP principles. IDH was applied undiluted (750 µL/ cornea, n=3). The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 137, and the mean in vitro irritancy score of the negative control (physiological saline) was 2.4. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. IDH did not induce ocular irritation through opacity or permeability, resulting in a mean in vitro irritancy score of -1.7 after 240 minutes of treatment. Since IDH induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).