Registration Dossier

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Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 11 April 2017 and 11 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 431 : In vitro Skin Corrosion: Human Skin Model Test and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: BZA
Chemical Name: Benzyl acrylate
CAS No.: 2495-35-4
Batch: 1B182601
Purity: 99.86%
Appearance: Colorless liquid
Expiry Date: 06 March 2018
Storage Conditions: At room temperature, under protection of sunlight
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Source strain:
not specified
Details on test system:
Epi-200 Kit Components Needed for the Assay
EpiDerm™ Kit Lot No.: 25811
1 Sealed 24-well plate Contains 24 inserts with EpiDerm™ tissues on agarose
2 24-well plates For MTT viability assay
4 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium DMEM-based medium
1 bottle DPBS Rinse Solution For rinsing the inserts in MTT assay

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant Solution (Isopropanol) For extraction of formazan crystals
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test Item Preparation
50 µL (79.4 µL/cm^2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surface
Duration of treatment / exposure:
3 minutes and 60 Minutes
Number of replicates:
2, duplicate tissues were treated with: test substance, positive control or negative control.
Irritation / corrosion parameter:
other: Relative absorbance
Run / experiment:
3 Minute exposure
Value:
100.4
Negative controls validity:
valid
Remarks:
Set to 100%
Positive controls validity:
valid
Remarks:
21.4
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: Relative absorbance
Run / experiment:
1 hour incubation
Value:
89.5
Negative controls validity:
valid
Remarks:
Set to 100%
Positive controls validity:
valid
Remarks:
4.3%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour

The test item is considered to be non-corrosive to skin:
• since the viability after 3 minutes exposure is greater than 50% and
• the viability after 1 hour exposure is greater than 15%


The acceptance criteria are met:
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.412 to 1.455)
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (4.3%)
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 1.0% to 9.8%)



Results after treatment with BZA and the controls

Dose Group

Ex-posure Inter-val

Absor-bance
Well 1
(Tissue 1/2)

Absor-bance
Well 2 (Tissue 1/2)

Absor-bance
Well 3 (Tissue 1/2)

Mean Absor-bance (Tissue 1/2)

Mean Ab-sorbance (OD) of 3 Wells minus Blank

Mean Ab-sorbance (OD) of 2 Tissues

Absor-bance [% of Negative Control]*

CV
[%]

Rel. Absor-bance [% of Negative Control]*

Blank

 

0.037

0.037

0.036

0.037

0.000

 

Negative Control

3
minutes

1.355

1.484

1.467

1.435

1.399

1.409

99.3

1.0

100.0

1.437

1.443

1.486

1.455

1.419

100.7

Positive Control

0.362

0.359

0.354

0.358

0.322

0.301

22.8

9.8

21.4

0.316

0.312

0.322

0.317

0.280

19.9

Test Item

1.410

1.465

1.493

1.456

1.419

1.421

100.8

0.2

100.9

1.455

1.463

1.462

1.460

1.424

101.1

Blank

 

0.036

0.036

0.035

0.035

0.000

 

Negative Control

1
hour

1.467

1.448

1.441

1.452

1.417

1.397

101.4

2.0

100.0

1.405

1.428

1.403

1.412

1.377

98.6

Positive Control

0.099

0.103

0.102

0.101

0.066

0.060

4.7

13.4

4.3

0.091

0.089

0.089

0.090

0.054

3.9

Test Item

1.162

1.156

1.145

1.154

1.119

1.133

80.1

1.8

81.1

1.188

1.187

1.174

1.183

1.147

82.2

*             relative absorbance [rounded values]:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The test item is considered to be non-corrosive to skin:

•       since the viability after 3 minutes exposure is greater than 50% and

•       the viability after 1 hour exposure is greater than 15%

The acceptance criteria are met:

•       the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.412 to 1.455)

•       the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (4.3%)

•       the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 1.0% to 9.8%)

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU CLP and UN GHS
Conclusions:
In conclusion, it can be stated that in this study and under the reported experimental conditions, BZA is non corrosive to skin according to EU CLP and UN GHS.
Executive summary:

This in vitro study was performed to assess the corrosive potential of BZA by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour, when mixed with deionised water (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the relative absorbance value did not decrease (100.4%) after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 89.5%. Both values did not affect the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item BZA is non corrosive to skin according to EU CLP and UN GHS.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
no
Specific details on test material used for the study:
Not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Not specified
Type of coverage:
occlusive
Preparation of test site:
not specified
Vehicle:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):0.5 mL
Duration of treatment / exposure:
4h
Observation period:
1, 24, 48 and 72 hours after removal of the patches
Number of animals:
3
Details on study design:
TEST SITE
- Type of wrap if used: lint patch and elastic medical tape

OBSERVATION TIME POINTS: 1, 24, 48 and 72h
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
72 h
Score:
2.9
Reversibility:
not reversible
Irritant / corrosive response data:
Well defined erythema (score 2) was observed in 3 rabbits from 1 to 72 hours
Very slight edema (score 1) and slight edema (score 2) were observed in animals 1 and 2, respectively at one hour after removal of patch.
Very slight edema (score 1) was observed in 3 rabbits from 24 to 48 hours after removal
At 72 hours after removal the edema disappeared in 1 animal, but very slight edema (score of 1) was still observed in 2 animals.
Interpretation of results:
Category 3 (mild irritant) based on GHS criteria
Conclusions:
From results described the test item was judged to be a moderate irritant to the rabbit skin under the conditions of this study.

Primary Dermal Irritation Index (P.D.I.I.) was calculated as 26/9 = 2.9
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 11 April 2017 and 02 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 : In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: BZA
Chemical Name: Benzyl acrylate
CAS No.: 2495-35-4
Batch: 1B182601
Purity: 99.86%
Appearance: Colorless liquid
Expiry Date: 06 March 2018
Storage Conditions: At room temperature, under protection of sunlight
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
Source strain:
not specified
Details on test system:
EpiSkin™ Kit Components Needed for the Assay

EpiSkin™ Kit Lot No.: 17-EKIN-022
1 Sealed 12-well plate Contains 12 inserts with EpiSkin™ tissues on agarose
1 12-well plate For MTT viability assay
1 bottle Assay Medium Basic medium for use in MTT assays
1 bottle EpiSkin™ Maintenance Medium Basic medium for incubations

MTT-Solution
The MTT-solution was prepared freshly on day of use with assay medium (concentration: 0.3 mg/mL).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test Item Preparation
10 µL (26.3 µL/cm^2) of the undiluted test item were applied to each of the triplicate tissues
Duration of treatment / exposure:
15 minutes
Number of replicates:
3, triplicate tissues were treated with test substance, positive control or negative control.
Details on study design:
MTT-Solution
The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).
For use in the pre-test (step 3): MTT from Sigma, Germany, DMEM from Gibco, Germany
For use in the main experiment: MTT concentrate from MatTek, MTT diluent from MatTek.

Cell Culture
Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diam).
EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 10 May 2017. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

Test for Direct MTT Reduction and Colour Interference
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility should be performed (step 1).
Step 1
50 µL of the test item were added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue. An additional test on viable tissues (without MTT addition) should be performed (step 2).
Since the test item did not dye water when mixed with it, step 2 did not have to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 50 µL of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item reduces MTT and an additional test on freeze-killed tissues (step 4) must be performed.
Since the test item did not prove to be a MTT reducer, step 4 did not have to be performed.

Experimental Performance
Pre-warming of EpiDerm™ Tissues
One hour before dosing, EpiDerm™ tissues were removed from the refrigerator, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Treatment
Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes
After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed. 

MTT Assay
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 µL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for 17.6 hours without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
3 x 200 µL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

Data Recording
The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item, negative, and positive controls.

Evaluation
The mean OD of the duplicate negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:

Relative viability (%) = (Mean OD test item/positive control / mean OD negative control) x 100

Interpretation of Results
For the test item and the positive control the mean relative viability  rel. standard deviation of the two individual tissues for both exposure periods was calculated and used for classification according to the following prediction model:
Viability measured after exposure time points Prediction to be considered
< 50% after 3 minutes exposure Corrosive
≥ 50% after 3 minutes exposure AND
< 15% after 60 minutes exposure Corrosive
≥ 50% after 3 minutes exposure AND
≥ 15% after 60 minutes exposure Non-corrosive

Test Item Identified as Corrosive
< 25% after 3 minutes exposure Optional Sub-category 1A*
≥ 25% after 3 minutes exposure A combination of optional Sub-categories 1B and 1C

* According to the data generated in view of assessing the usefulness of the RhE test methods for supporting sub-categorisation, it was shown that around 29%, 31% and 33% of the Sub-category 1A results of the EpiDERMTM test method may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications).

Acceptability of the Assay
An assay met the acceptance criteria if
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30%
The quality certificate of the supplier of the test kit demonstrating its robustness (treatment with 1% Triton X-100: 4.77 hours ≤ ET50 ≤ 8.72 hours) is annexed to the report.

Irritation / corrosion parameter:
other: Relative absorbance
Run / experiment:
15 Minute exposure
Value:
6.9
Negative controls validity:
valid
Remarks:
Set to 100%
Positive controls validity:
valid
Remarks:
5.3
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 6.9% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

The acceptance criteria were met:
• the mean OD of the three negative control exposed tissues is ≥ 0.6 till ≤ 1.5 (range: 0.856 to 0.972).
• the rel. standard deviations between tissues of the same treatment group was ≤ 18% (range:3.8 to 12.7%).
• the mean relative tissue viability of the positive control was ≤ 40% (5.3%). • the acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.7 mg/mL).
• the results for the positive control and negative control (deionized water; data for PBS are in process) are within the historical data (means, rel. standard deviation, and ranges) of Envigo CRS GmbH

Results after treatment with BZA and the controls

Dose Group

Absorbance 570nm
Tissue

Well 1

Absorbance 570 nm
Tissue

Well 2

Mean Absorbance 570nm

Mean Absorbance

570 nm

*

Mean Absorbance (%) of 3 Tissues

Relative Absorbance [%] Tissue 1, 2 + 3

**

Relative Standard Deviation
[%]

Rel. Absorbance [% of Negative Control]**

 Blank  0.038  0.038  0.38  0.000        
 Negative Control  0.945  0.905  0.925  0.887  0.880  100.8  6.6  100.0
   0.983  0.960  0.972  0.934    106.2    
   0.872  0.840  0.856  0.818    93.0    
 Positive Control  0.073  0.083  0.078  0.040  0.046  4.5  12.7  5.3
   0.091  0.080  0.086  0.048    5.4    
   0.087  0.091  0.089  0.051    5.8    
 Test Item  0.102  0.099  0.100  0.062  0.061  7.1  3.8  6.9
   0.097  0.096  0.096  0.058    6.6    
   0.099  0.102  0.102  0.063    7.1    

* Mean of two replicate wells after blank correction

** relative absorbance per tissue [rounded values]:  

*** relative absorbance per treatment group [rounded values]:  

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.  

Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 6.9% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

The acceptance criteria were met:

• the mean OD of the three negative control exposed tissues is ≥ 0.6 till ≤ 1.5 (range: 0.856 to 0.972).

• the rel. standard deviations between tissues of the same treatment group was ≤ 18% (range: 3.8 to 12.7%).

• the mean relative tissue viability of the positive control was ≤ 40% (5.3%).

• the acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.7 mg/mL).

• the results for the positive control and negative control (deionized water; data for PBS are in process) are within the historical data (means, rel. standard deviation, and ranges) of Envigo CRS GmbH

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU CLP and UN GHS
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, BZA is irritant to skin according to UN GHS and EU CLP regulation
Executive summary:

This in vitro study was performed to assess the irritation potential of BZA by means of the Human Skin Model Test according to OECD TG 439.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water, when mixed with it (pre-test for colour interference). Consequently, additional tests with freeze-killed tissues or viable tissues (without MTT addition) were not necessary.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased to 6.9%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, BZA is irritant to skin according to UN GHS and EU CLP regulation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted on 12 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and in compliance with GLP.
Qualifier:
according to
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 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: BZA
Chemical Name: Benzyl acrylate
CAS No.: 2495-35-4
Batch: 1B182601
Purity: 99.86%
Appearance: Colorless liquid
Expiry Date: 06 March 2018
Storage Conditions: At room temperature, under protection of sunlight
Stability in Solvent: Not indicated by the Sponsor
Purpose of Use: Industrial chemical
Species:
other: Eyes from adult cattle
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
Collection of Bovine Eyes Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the cornea.
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 Minutes
Number of animals or in vitro replicates:
3 corneas per treatment
Details on study design:
Experimental Design and Study Conduct

Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

Exposure of the Corneae to the Test Groups
The corneae were distributed as follows:
Groups Number of Corneae
1 Negative Control 3
2 Positive Control 3
3 Test Item 3

The anterior compartment received the test item or negative or positive control at a volume of 0.75 mL on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes. After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). The opacity measurement is described below.
In the second step of the assay, permeability of the corneae was determined. The permeability measurement is described below.

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 Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groups with the test item, negative, and positive controls.

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 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.
Irritation parameter:
in vitro irritation score
Value:
> 1.56 - < 2.69
Negative controls validity:
valid
Remarks:
1.23
Positive controls validity:
valid
Remarks:
76.41
Remarks on result:
no indication of irritation

Results after 10 Minutes Incubation Time


Test Group

Opacity value = Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0

0.078

0.082

1.17

1.23

Not categorized

0

0.082

1.23

0

0.085

1.28

Positive Control

65.00*

0.794*

76.92

76.41

Category 1

70.00*

0.696*

80.45

62.00*

0.658*

71.88

Benayl Acrylate (BZA)

2.00*

-0.030*

1.56

2.14

Not categorized

2.00*

-0.011*

2.17

3.00*

-0.021*

2.69

* corrected values

Interpretation of results:
other: Non-irritating, not requiring classification
Remarks:
Criteria used for interpretation of results: EU CLP and UN GHS.
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, BZA is not categorized (GHS).
Executive summary:

This in vitro study was performed to assess the corneal damage potential of BZA by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity 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 (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).  

Relative to the negative control, the test item BZA did not cause a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 2.14. According to OECD 437 (see table in chapter 3.8.3) the test item is not categorized (GHS).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification