Prop-2-yn-1-yl 1H-imidazole-1-carboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 26. Jun. 2020

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:

adopted 14. Feb. 2017

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160: “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY AND ISOLATED CHICKEN EYE TEST METHODS”

Version / remarks:

25. Oct. 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-yn-1-yl 1H-imidazole-1-carboxylate

EC Number:

695-595-2

Cas Number:

83395-38-4

Molecular formula:

C7H6N2O2

IUPAC Name:

prop-2-yn-1-yl 1H-imidazole-1-carboxylate

Test material form:

solid

Specific details on test material used for the study:

Name: 2-propyn-1-yl 1H-imidazole-1-carboxylate
Batch no.: 0802-2#
CAS no.: 83395-38-4
Composition: 2-propyn-1-yl 1H-imidazole-1-carboxylate
Storage: fridge (2 - 8 °C) under inert gas
Expiry date: 11. Jul. 2021
Stability : stable under storage conditions
Appearance: white solid
Purity: 99.1 %
Homogeneity: homogeneous
Production date: 13. Oct. 2020
EC no.: 695-595-2
Molecular formula: C7H6N2O2
Molecular weight: 150.13 g/mol

Test animals / tissue source

Species:

other: Bovine

Strain:

other: Species Bos primigenius Taurus (fresh bovine corneas)

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Indus-triestraße 42, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour.

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):
Test item1: 305.3 mg; Test item2: 305.5 mg; Test item3: 303.9 mg
Positive control: 750 μL
Negative control: 750 μL

- Concentration (if solution):
Test item: Tested directly, without dilution or preparation of a solution
Positive control: Imidazole solution: 20% C3H4N2 (CAS-No. 288-32-4), dissolved in HBSS, batch no.: T20210225
Negative control: HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in de-min. water (1:10), batch no.: T20210225

Duration of treatment / exposure:

4 hours at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

90 minutes at 32 ± 1 °C

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS :
After having carefully cleaned and sterilized the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outer edges. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incu-bated for 1 hour in the incubation chamber at 32 ± 1 °C. The formation of bubbles was prevented.

QUALITY CHECK OF THE ISOLATED CORNEAS :
The corneas were examined visually and only corneas which were free from damages were used.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : Yes (HBSS)

POSITIVE CONTROL USED : Yes (20% imidazole solution)

APPLICATION DOSE AND EXPOSURE TIME: 750 μL, 4 hours at 32 ± 1 °C

TREATMENT METHOD: Closed Chamber Method for controls; Open Chamber Method for the test item

POST-INCUBATION PERIOD: yes, 90 minutes at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change of opacity value of each treated cornea with test item, positive control or neg-ative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea.
The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [microtiter plate reader] (OD492)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

ca. 139.08

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	1.06	1.06	13.58%
	1.21
	0.92
Test Item 2-propyn-1-yl 1H-imidazole-1-carboxylate	164.52	139.08	15.85%
	127.28
	125.45
Positive Control 20% imidazole solution	112.08	102.88	9.25%
	103.48
	93.08

The validity criteria and findings are given in the following table:

Parameter	Criterion	Found	Assessment
Mean IVIS of negative control HBSS	<=3	1.06	ok
Mean IVIS of positive control 20% imidazole solution	75.21 – 143.42	102.88	ok

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the conditions of this study, the test item 2-propyn-1-yl 1H-imidazole-1-carboxylate induced serious eye damage on the cornea of the bovine eye. The calcu-lated mean IVIS was 139.08.
According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS > 55 in-duces serious eye damage, that should be classified as UN GHS Category I.

Executive summary:

This in vitro study was performed to assess the corneal damage potential of 2-propyn-1-yl 1H-imidazole-1-carboxylate by quantitative measurements of changes in opacity and permeability in a bovine cornea.

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item2-propyn-1-yl 1H-imidazole-1-carboxylatewas applied onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated mean IVIS (In VitroIrritancy Score) was 1.06.

20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated mean IVIS was 102.88.

Under the conditions of the test, the test material is considered to be induces serious eye damage.