4-isopropyl-m-cresol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 15, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD test Guideline No. 437 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)

Test material

Test material information

Constituent 1

Reference substance name:

4-isopropyl-m-cresol

EC Number:

221-761-7

EC Name:

4-isopropyl-m-cresol

Cas Number:

3228-02-2

Molecular formula:

C10H14O

IUPAC Name:

4-isopropyl-m-cresol

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Parathymol P16029
- Physical state: White crystalline solid
- Storage condition of test material: Room temperature in the dark

Test animals / tissue source

Species:

other: Bovine eye

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- 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 mg/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:

other: 0.9% w/v sodium chloride solution

Amount / concentration applied:

TEST MATERIAL
- Test Item Formulation and Experimental Preparation: For the purpose of this study the test item was prepared as a 20% w/v solution in 0.9% w/v sodium chloride solution. The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
- Amount(s) applied (volume or weight with unit): 0.75 mL was applied on each cornea

Duration of treatment / exposure:

Test item applied BCOP holder was incubated, anterior chamber uppermost at 32 ± 1 °C for 240 minutes.

Observation period (in vivo):

- Corneal opacity was measured pre-treatment, post-treatment and post-incubation (after 240 minutes of incubation).
- Application of Sodium Fluorescein: Following the opacity measurement the permeability of the corneas to sodium fluorescein (5 mg/mL) was evaluated and then corneal holders were incubated, anterior chamber uppermost at 32 ± 1 °C for 90 minutes.

Number of animals or in vitro replicates:

Total: 9 corneas - 3 corneas/group for test item, negative and positive controls

Details on study design:

Details of test procedure:
- Treatment of corneas: Corneas obtained from freshly slaughtered adult cattle (from a local abattoir) were mounted in corneal holders. Both chambers of each BCOP holder were filled with complete Eagle’s minimum essential medium (MEM) and incubated for 60 minutes at 32 ± 1 °C. Before the treatment, opacity measurement was performed using an opacitometer. MEM was removed from anterior chamber and the test item (0.75 mL) was applied on each cornea. The holders were incubated, anterior chamber uppermost, at 32 ± 1 °C 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 three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.
- Application of sodium fluorescein: 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 °C for 90 minutes.
- Permeability determinations: After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Negative control: 0.9 % w/v sodium chloride solution
Positive control: Imidazole, 20% w/v solution in 0.9 % w/v sodium chloride solution

Results and discussion

In vivo

Results

Irritation parameter:

other: In Vitro Irritancy Score (IVIS)

Basis:

mean

Time point:

other: 240 minutes

Score:

46.9

Max. score:

55

Irritant / corrosive response data:

- In Vitro Irritancy Score (IVIS) for test item, negative and positive controls were 46.9, 2.3 and 95.3, respectively.
- Corneal Epithelium Condition: The corneas treated with the test item were cloudy with sloughing post treatment. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.
- The IVIS for the test item was 46.9. This score is below the cut-off for classification of serious eye damage set at > 55.
- In the three test item treated corneas sloughing of the corneal epithelium was observed. However in two of the three test item treated corneas the sloughing is considered to be more advanced resulting in lower opacity values (upper cloudy layers of the cornea have sloughed away revealing clearer layers underneath) The opacity was markedly higher in the cornea that had not sloughed as significantly. It is therefore apparent that if the sloughing was not as advanced in the first two corneas the overall IVIS would have been higher.
- In addition all three test item treated corneas induced fluorescein permeability values greater than noted in the three positive control corneas.

Other effects:

None

Any other information on results incl. tables

The post treatment condition of the corneal epithelium was as follows:

 

Treatment	Epithelium Condition
Test Item	Clear in three corneas
Negative Control	Cloudy in three corneas
Positive Control	Cloudy & sloughing in three corneas

 

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 73.8 - 103.6. The positive control acceptance criterion was therefore satisfied.

 

The negative control gave opacity of ≤ 4.7 and permeability ≤ 0.085. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the test conditions, Parathymol is classified as Category 1, H318 Causes serious eye damage according to the Regulation (EC) No. 1272/2008.

Executive summary:

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, Parathymol was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution 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 anIn VitroIrritancy Score (IVIS).

 

In Vitro Irritancy Score (IVIS) for test item, negative and positive controls were 46.9, 2.3 and 95.3, respectively. In the three test item treated corneas sloughing of the corneal epithelium was observed. However in two of the three test item treated corneas the sloughing was considered to be more advanced resulting in lower opacity values (upper cloudy layers of the cornea have sloughed away revealing clearer layers underneath.) The opacity was markedly higher in the cornea that had not sloughed as significantly. It is therefore apparent that if the sloughing was not as advanced in the first two corneas the overall IVIS would have been higher. In addition all three test item treated corneas induced fluorescein permeability values greater than noted in the three positive control corneas.

 

Under the test conditions,Parrathymol is classified as Category 1, H318 Causes serious eye damage according to the Regulation (EC) No. 1272/2008.