4-chlorobenzoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.05.21 – 20.05.21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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:

according to OECD Test Guideline No. 437 (2017)

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 14th February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-chlorobenzoic acid

EC Number:

200-805-9

EC Name:

4-chlorobenzoic acid

Cas Number:

74-11-3

Molecular formula:

C7H5ClO2

IUPAC Name:

4-chlorobenzoic acid

Test material form:

solid: particulate/powder

Details on test material:

white to yellowish

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). The results were based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay were from the same group of eyes collected on a specific day.

Selection criteria for eyes used in BCOP:
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularization. Only corneas from eyes free of such defects were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.
From 25 eyes, 6 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 1, 2, 3, 4, 5, 6, 7, 8, and 9), 7 corneas were superfluous and 3 corneas were used for testing of other items.

Number of corneas per group:
Exposed group (test item) - 3 corneas (No. 7, 8, 9)
Positive control group (20% w/v imidazole in 0.9% NaCl solution) – 3 corneas (No. 4, 5, 6)
Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

47.75

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corrected mean opacity

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

54.57

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

60.31

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

3

Value:

46.13

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The study met all the acceptance criteria for positive and negative controls.

Any other information on results incl. tables

Calculation of IVIS

 

The In Vitro Irritancy Score (IVIS) was computed according the following formula:

 

IVIS = mean opacity value + (15 x mean permeability OD₄₉₀value)

 

 IVIS values

Group	IVIS
	Calculation	Result
NC (0.9% NaCl)	1.13 + (15 x 0.0013)	1.15
PC (20% Imidazole in 0.9% NaCl)	43.47 + (15 x 1.8657)	71.46
Test item (4-chlorobenzoic acid)	47.75 + (15 x 0)	47.75

Note: NC- negative control, PC- positive control

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The study met all the acceptance criteria for positive and negative controls. The In Vitro Irritancy Score (IVIS) for test item, 4-chlorobenzoic acid was 47.75.

This value of IVIS is >3 and simultaneously ≤55 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no stand-alone prediction can be made.

Executive summary:

The test item, 4-chlorobenzoic acid, was tested for the potential to cause ocular corrosivity or severe irritancy, as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to OECD Test Guideline No. 437 (2017) and Method B.47 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, Commission Regulation (EU) 2017/735, Adopted 14^thFebruary 2017.

The testing was performed on three groups of corneas: test item treatment group, positive control group (20% w/v imidazole in 0.9% NaCl solution) and negative control group (0.9% NaCl solution). Three corneas per group were used.

Open-chamber method was used, because the test item was not soluble in water or 0.9% NaCl. The test substance was applied in 100 % concentration.

The opacity and permeability of each cornea were measured.The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

The study met all the acceptance criteria for positive and negative controls. The In Vitro Irritancy Score (IVIS) fortest item, 4-chlorobenzoic acid was 47.75.

This value of IVIS is >3 and simultaneously ≤55 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no stand-alone prediction can be made.