Butyl anthranilate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

other information

Study period:

The test was performed on 05 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 25 April 2012, 23 ,25,Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

Butyl anthranilate

EC Number:

231-816-7

EC Name:

Butyl anthranilate

Cas Number:

7756-96-9

Molecular formula:

C11H15NO2

IUPAC Name:

butyl 2-aminobenzoate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Anthranilsäurebutylester

Specific details on test material used for the study:

Identification: Anthranilsäurebutylester
Batch: 1/12
CAS No.: 7756-96-9
EINECS: 231-816-7
Purity: 99.9%
Appearance: Bright yellow clear liquid
Expiry Date: 02 May 2018
Storage Conditions: At room temperature
Stability in Solvent: Not indicated by the Sponsor
Certificate of Analysis: AZ 1014/Toxd1

Test animals / tissue source

Species:

other: Freshly isolated bovine cornea (at least 9 month old donor cattle)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was tested undiluted.
Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

10 minutes

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of the test item, the negative, and the positive control for 10 minutes.

SCORING SYSTEM:
Opacity Measurement:
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

Permeability Determination:
Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Data Evaluation

Opacity:
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0).
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability:
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.


IVIS Calculation:

The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following category according to OECD guideline 437:
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Results and discussion

In vivo

Irritant / corrosive response data:

Relative to the negative control, the test item Anthranilsäurebutylester did not cause any increase of the corneal opacity or permeability. The calculated mean IVIS was 1.08 (threshold for No Category: IVIS ≤ 3).
According to OECD 437 the test item is identified as not requiring classification for eye irritation or serious eye damage i.e. No Category (UN GHS).

Any other information on results incl. tables

Results after 10 Minutes Incubation Time

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	ProposedUN GHS Category
		Mean		Mean
Negative Control	0	0.00	0.060	0.053	0.90	0.80	No Category
	0		0.050		0.75
	0		0.050		0.75
Positive Control	90.00*		1.046*		104.89	100.50	Category 1
	83.00*		1.404*		103.26
	79.00*		1.009*		93.34
Test Item	0.00*		-0.004*		-0.07*	1.08	No Category
	1.00*		0.017*		1.25*
	2.00*		0.003*		2.04*

*corrected values

Applicant's summary and conclusion

Interpretation of results:

other: The calculated mean IVIS was 1.08 (threshold for No Category: IVIS ≤ 3). According to OECD 437 the test item is identified as not requiring classification for eye irritation or serious eye damage i.e. No Category (UN GHS).

Conclusions:

according to the current study and under the experimental conditions reported, Anthranilsäurebutylester is identified as not inducing eye irritation or serious eye damage

Executive summary:

This in vitro study was performed to assess thecorneal damage potential of Anthranilsäurebutylester by means of the BCOP assay usingfresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the neat test item Anthranilsäurebutylester, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae wereincubated for another 120 minutes at 32 ± 1 °C in incubation medium, andopacity was measured a second time (t₁₃₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS= 0.80).

The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (meanIVIS =100.50)corresponding to a classification asserious eye damaging (CLP/EPA/GHS (Cat 1)).

The test item Anthranilsäurebutylester was tested undiluted.Relative to the negative control, the test item Anthranilsäurebutylester did not cause any increase of the corneal opacityor permeability.The calculated mean IVIS was 1.08 (threshold for No Category: IVIS≤ 3). According to OECD 437 the test itemis identified as not requiring classification for eye irritation or serious eye damage i.e. No Category (UN GHS).