Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-April-2021 to 15-April-2021 (experimental dates)

Reliability:

1 (reliable without restriction)

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:

26 June 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)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance Document on ‘The Collection of Tissues for Histological Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160.

Version / remarks:

6 July 2018 (Paris)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Test material form:

solid: particulate/powder

Details on test material:

Appearance: beige powder

Specific details on test material used for the study:

Appearance: Pale yellow solid
Storage conditions: Room temperature in the dark

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test item: An attempt was made to formulate the test item as a 20% w/v solution in sodium chloride 0.9% w/v but was found to be not suitable for dosing. Therefore, the test item was applied neat. A disc of test item was formed to cover the whole cornea. Each disc weighed approximately 0.3678 g

Control: 0.75 mL

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

1.5 hours

Number of animals or in vitro replicates:

3 replicates/group

Details on study design:

Preparation and selection of corneas:
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative, positive control and the test item experiment.

Treatment of Corneas:
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. The test item would not form a suitable suspension for dosing therefore approximately 0.3678 g of the solid test item was found to adequately cover the corneal surface. 0.75ml of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Run 2

Value:

0

Negative controls validity:

valid

Remarks:

1.6

Positive controls validity:

valid

Remarks:

81.3

Other effects / acceptance of results:

The test was performed twice (runs 1 & 2) as the acceptance criteria was not met for the negative control item in the first run. The corneal epithelia appeared clear for the test item and the control treatments, the cornea in the positive control group all appeared cloudy after treatment.
The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied in run 2.
The negative control gave opacity and permeability values within an accepted range. The negative control acceptance criteria were therefore considered to have been satisfied in run 2.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this bovine corneal opacity permeability (BCOP) assay (OECD 437) and according to UN GHS classification, the test item BTMR is given No Category classification under the conditions of the test.

Executive summary:

This this bovine corneal opacity permeability (BCOP) assay was carried out according to the OECD 437 guideline.

The test item was applied neat to the cornea for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The in vitro irritation scores for BTMR was found to be 0.0. The negative and positive control irritation score was of 1.6 and 81.3, respectively. 

Under the conditions of this bovine corneal opacity permeability (BCOP) assay (OECD 437) and according to UN GHS classification, the test item BTMR is given No Category classification under the conditions of the test.

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