D,L- Menthol / D,L-Isomenthol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2018 to 02 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

(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:

(2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

D,L- Menthol / D,L-Isomenthol

Molecular formula:

C10H20O

IUPAC Name:

D,L- Menthol / D,L-Isomenthol

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: Slaughterhouse (AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany)
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): 9-month-old cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in HBBS containing 1 % (v/v) Penicillin/Streptomycin in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Not specified
- indication of any existing defects or lesions in ocular tissue samples: All eyes were checked for unacceptable defects by removing them from the storage solution and examining them. Those exhibiting defects were discarded.
- Indication of any antibiotics used: The cattle eyes were stored in HBSS containing 1% Penicillin/Streptomycin during transportation.

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): 0.75 ml was introduced onto the epithelium of the cornea.
- Concentration (if solution): The test material was prepared as a 20 % suspension (w/v) in saline.

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% NaCl
- Lot/batch no. (if required): Not specified
- Purity: Not specified

Duration of treatment / exposure:

The test consisted of the application of the 20 % (w/v) suspension in saline of the test item to the fresh bovine cornea; followed by an initial opacity measurement. After exposure, the cornea was thoroughly rinsed to remove the test item and incubated for 240 minutes in incubation medium. After incubation in the medium, opacity measurements and permeability values were determined after a 90 minutes incubation period in sodium fluorescein.

Duration of post- treatment incubation (in vitro):

90 +- 5 minutes at 32 +-1°C

Number of animals or in vitro replicates:

Three test replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Preparation: The eyes were checked for defects such as opacity, scratches, pigmentation and vascularization by removing them from the HBSS containing 1 % (v/v) Penicillin/Streptomycin.


QUALITY CHECK OF THE ISOLATED CORNEAS : All eyes were checked macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

NUMBER OF REPLICATES : Three

NEGATIVE CONTROL USED : Physiological saline

SOLVENT CONTROL USED (if applicable) : Not applicable
POSITIVE CONTROL USED : 10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl (saline)

APPLICATION DOSE AND EXPOSURE TIME : 0.75 ml was introduced onto the epithelium of the cornea.

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: 90 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Not specified
- POST-EXPOSURE INCUBATION: 240 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a Opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Not specified

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
In vitro score range UN GHS
≤ 3 No Category
> 3 ≤ 55 No prediction can be made
>55 Category 1


DECISION CRITERIA: The decision criteria for the IVIS cut-off values for identifying test chemicals as inducing serious eye damage was used for the study.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item Mean

Value:

21.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not applicable

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The first negative control value was outside the acceptance criteria range of 0.86 – 1.64. Due to this, the experiment was repeated twice. The overall IVIS mean for the negative control was 1.03, which was within the acceptance criteria range.
- Acceptance criteria met for positive control: The positive control value of 108.55 was within the acceptance criteria range of 98.30 – 138.03.

Any other information on results incl. tables

Table 1: results after 240 minutes exposure period.

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean		1.03	No category
Negative control	1	0.33	0.045	0.046	1.68
	0		0.044		0.66
	0		0.050		0.75
Positive control	95.67*		0.627*		105.07	108.55	Category 1
	103.67*		0.735*		114.69
	10.67*		0.483*		105.91
Test item	8.67*		0.964*		23.12	21.33	No prediction can be made
	7.67*		0.880*		20.86
	10.67*		0.623*		20.01

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Not corrosive or a severe irritant to the eye but study is not sufficient for hazard assessment

Conclusions:

In conclusion, under the reported experimental conditions the test item did not cause serious eye damage (Category 1: CLP/GHS). However, the study is not sufficient to be used for hazard classification under the CLP regulation.

Executive summary:

The study is a GLP-compliant study following OECD guideline 437 and is therefore reliable without restrictions (Klimisch 1).  A 20 % (w/v) suspension in saline of the test item was applied directly on top of the corneas. The positive control mean in vitro irritancy score of 108.55 was within two standard deviations of the current historical positive control mean. The negative control values for opacity and permeability (1.03) were less than the upper limits of the laboratory historical range. The validity criteria for the study was therefore met. The test item had an In vitro Irritancy score of 21.33. Based on this result the test item did not cause serious eye damage (Category 1: CLP/GHS); however, this study is not sufficient to be used for hazard classification under CLP regulation.