Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP. There was an uncritical deviation from the guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. This can be seen as uncritical, because the opacity can be calculated from the absorbances.
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
yes
Remarks:
The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. This can be seen as uncritical, because the opacity can be calculated from the absorbances.
GLP compliance:
yes (incl. certificate)
Remarks:
(Date of inspection: 2014-10-13/14, Date of signature: 2015-04-08)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals / tissue source

Species:
other: Bovine
Strain:
not specified

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: Three corneas were used for positive and negative controls
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μl

CONTROLS
- Positive control substance: Dimethylformamide, DMF, CAS-No. 68-12-2, undiluted.
- Negative control substance: Sodium chloride solution: 0.9% NaCl (CAS-No. 7647-14-5), dissolved in demin. water.
Duration of treatment / exposure:
10 minutes incubation at 32 ± 1°C
Observation period (in vivo):
The corneas were stored for additional 2 h at 32 ± 1 °C (post-incubation).
Optical density measured after a further 90 ± 5 min at 32 ± 1 °C incubation in medium with fluoroscein
Number of animals or in vitro replicates:
For each treatment group (negative control solution, test item and positive control), three replicates were used.

Results and discussion

In vivo

Results
Irritation parameter:
other: In vitro irritation score
Basis:
other: opacity and permeability
Score:
0.02
Remarks on result:
other: negative control score: 0.21; positive control score: 105.86; not irritating

Any other information on results incl. tables

Opacity and Permeability Values      

For each treatment group (negative control solution, test item and positive control), three replicates were measured.

The absorbance (570 nm) and opacity values which were measured before and after exposure are given in the following table:

Absorbance and Opacity Values Negative Control

Parameter

Negative Control

Absorbance before exposure

0.1888

0.1907

0.1792

Absorbance after exposure

0.2395

0.2033

0.2148

Opacity before exposure

1.5445

1.5513

1.5108

Opacity after exposure

1.7358

1.5970

1.6398

Opacity Difference

0.1913

0.0457

0.1291

Mean opacity difference of the negative control is 0.1220.

 

Absorbance and Opacity Values Test Item and Positive Control

Parameter

Test Item
Alpha Methylenebenzyl Acetate            

Positive Control

Absorbance before exposure

0.3106

0.1407

0.2123

0.1425

0.1296

0.1766

Absorbance after exposure

0.3383

0.1515

0.3066

1.9991

1.8398

1.9957

Opacity before exposure

2.0446

1.3826

1.6304

1.3884

1.3477

1.5018

Opacity
after exposure

2.1792

1.4174

2.0258

99.7930

69.1512

99.0148

Opacity
Difference

0.1347

0.0348

0.3954

98.4046

67.8035

97.5130

 

 

 

For the permeability measurement, three replicates for each treatment group were measured. The optical density values at 490 nm are given in the following table:

Optical density at 490 nm

Replicate

Negative Control

Test ItemAlpha Methylenebenzyl Acetate

Positive Control

Measured values

-0.0004

0.0007

0.0031

0.0003

-0.0004

0.0016

0.1997

0.2629

0.2637

*Corrected values

-0.0020

0.0035

0.0155

0.0015

-0.0020

0.0080

0.9985

1.3145

1.3185

Mean

0.0057

--

*Note: In order to correct the path length, a factor of 5 was taken into account when calculating the IVIS (see previous chapter).


IVIS Values     

IVIS was calculated using the values in tables 9.1-a, 9.1-b and 9.1-c and the equation stated in chapter8.3.

Example:

IVIS (Test Item Alpha Methylenebenzyl Acetate, Repl. 1) =

(0.1347 – 0.1220) + [15 * (0.0015 – 0.0057)] = -0.05

 

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

IVIS

Test Group

IVIS

Mean IVIS

Standard Deviation of IVIS

Relative Standard Deviation of IVIS

Negative Control
0.9% NaCl

0.16

0.21

0.138

66.4%

0.10

0.36

Test Item
Alpha Methylenebenzyl Acetate

-0.05

0.02

0.262

1456.2%

-0.20

0.31

Positive Control
DMF undiluted

113.18

105.86

16.178

15.3 % 

87.31

117.08

 

Note: the high relative standard deviations of the IVIS of test item and negative control are due to mathematical reasons, as the respective means are very small.


 

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The eye irritancy potential of α-methylenebenzyl acetate has been investigated in a valid in vitro bovine corneal opacity and permeability assay conducted in accordance with OECD 437 and in compliance with GLP. The following mean in vitro irritation score was calculated: 0.02. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. It is concluded that the test substance is not irritating to the eye.
Executive summary:

α-Methylenebenzyl acetate has been tested for eye irritation investigated in a valid in vitro bovine corneal opacity and permeability assay conducted in accordance with OECD 437 and in compliance with GLP. The test item did not meet the criteria for classification.