α-methylenebenzyl acetate

Endpoint summary

Description of key information

In vitro skin and eye irritation studies found the test item to be non-irritating.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-05-19 to 2015-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Date of inspection: 2014-10-13/14, Date of signature: 2015-04-08)

Species:

other: EpiDerm

Vehicle:

unchanged (no vehicle)

Controls:

other: positive and negative control tissues

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 60 μl (0.63 μl/cm2)
- Concentration (if solution): undiluted

CONTROLS: Controls were set up in parallel to the test item cultures in order to confirm the validity of the test.
Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and without MgCl2).
Positive control: Sodium dodecyl sulphate (SDS), CAS No. 151-21-3, solution in deionised H2O containing 5% SDS. Procured from MatTek In Vitro Life Science Laboratories, batch no.: 020615MHG

Duration of treatment / exposure:

60 minutes

Observation period:

Treated tissue had a total incubation time of 42 h

Number of animals:

Three tissues for each of test, negative control and positive control groups

Details on study design:

TEST SYSTEM:
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized 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 EpiDermTM tissues are cultured on specially prepared.

PRE-EXPERIMENT
The test item was tested for the ability of direct MTT reduction. To test for this ability, 30 µL of the liquid test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5 ± 0.5% CO2 for 60 min. Untreated MTT solution was used as control.
The MTT solution didn’t change its colour within one hour, therefore, direct MTT reduction had not taken place, and no data correction was necessary


EXPERIMENTAL PROCEDURE

Pre-Incubation of Tissues
Eight 6-well-plates were prepared with 0.9 mL assay medium in three of the six wells (upper row). The tissues were inspected for viability. Viable tissues were transferred (three per plate) in the wells with the medium using sterile forceps under the clean bench and placed into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 1 h.
After the pre-incubation (one hour), the other three wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 18 h.

Treatment
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only.
One plate (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used as positive control; each tissue was treated with 30 µL SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used for treatment with the test item:
30 µL test item were applied and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in one minute intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 min. 37 ± 1°C and 5 ± 0.5% CO2. 60 min. after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in one-minute-intervals.
After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 24 h.

Medium Renewal
For three incubated tissues, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for 10 min. (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the incubator for 18 h for post-incubation.

MTT Assay
After a total incubation time of 42 h, a 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 h.
After this time, the MTT reagent was aspirated and replaced by DPBS buffer. This was also then aspirated and replaced several times.
At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 h at room temperature.
After 2 h, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, 2 replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

Irritation / corrosion parameter:

other: other: tissue viability

Value:

122.9

Remarks on result:

other:

Remarks:

Basis: other: mean optical density relative to control. Remarks: not irritating . (migrated information)

Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Absorbance values blank isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.038	0.037	0.037	0.038	0.036	0.037	0.037	0.037	0.037

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values negative control, test item and positive control (OD at 570 nm)

Designation	Measurement	Negative Control	Alpha Methylenebenzyl Acetate	Positive Control
Tissue 1	1	1.828	2.240	0.088
	2	1.794	2.250	0.085
Tissue 2	1	1.786	2.191	0.083
	2	1.780	2.212	0.083
Tissue 3	1	1.877	2.233	0.089
	2	1.840	2.228	0.089

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in the table above. Mean and relative standard deviation (comparison of the 3 tissues) were also calculated.

Mean Absorbance Values

Designation	Negative Control	Alpha Methylenebenzyl Acetate	Positive Control
Mean – blank (tissue 1)	1.774	2.208	0.050
Mean – blank (tissue 2)	1.746	2.165	0.046
Mean – blank (tissue 3)	1.822	2.194	0.052
Mean of the 3 tissues	1.781	2.189	0.049
Relative standard deviation of the 3 tissues	2.2%	1.0%	6.2%

 

Comparison of Formazan Production

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table9.2‑a           % Formazan Production

Designation	Alpha Methylenebenzyl Acetate	Positive Control
% Formazan production (tissue 1)	124.0%	2.8%
% Formazan production (tissue 2)	121.6%	2.6%
% Formazan production (tissue 3)	123.2%	2.9%
% Formazan production (mean)	122.9%	2.8%

 

Assessment and Validity

Skin Irritation Potential of the Test Item

The relative absorbance values were increased to 122.9 % after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as not irritant to skin.

Validity and Acceptability

Validity criteria and results are stated in the following table:

Criterion	Demanded	Found
OD of negative control	≥0.8 and≤ 2.8	1.8
% Formazan production of positive control SDS	<20% of negative control	2.8 %
Variation within replicates (RSD)	< 18%	2.2 % (negative control) 6.2 % (positive control) 1.0 % (test item)

 

All validity criteria were met.

Values for negative control and for positive control were within the range of historical data of the test facility.

Therefore, the experiment is considered valid.

 

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

α-Methylenebenzyl acetate has been tested in a reliable in vitro study for skin irritation conducted according to OECD 439 and in compliance with GLP using EpiDerm tissue. The relative tissue viability of the test-item treated tissues, was not reduced relative to the negative controls. The positive control produced the expected reduction in viability. It is concluded that the test item is not irritant under the conditions of the test.

Executive summary:

α-Methylenebenzyl acetate has been tested for skin irritation in a study carried out according to OECD Guideline 439, compliant with GLP.

Following a 60 minute application of the test substance to the human skin model, it was concluded that the test item is not irritating to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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

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.

Qualifier:

according to guideline

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

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. QA statement)

Remarks:

(Date of inspection: 2014-10-13/14, Date of signature: 2015-04-08)

Species:

other: Bovine

Strain:

not specified

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.

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

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.

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

α-Methylenebenzyl acetate has been tested for skin corrosion in an in vitro study conducted according to OECD Guideline 431, and in compliance with GLP (LAUS, 2015). Following 3 minute and 1 hour applications of the test substance to the human skin model EpiDerm it was concluded that the test substance was non-corrosive. 

 

α-Methylenebenzyl acetate has been tested in a reliable in vitro study for skin irritation conducted according to OECD 439 and in compliance with GLP using EpiDerm tissue (LAUS, 2015). The relative tissue viability of the test substance treated tissues, was not reduced relative to the negative controls. The positive control produced the expected reduction in viability. It is concluded that the test substance is not irritant under the conditions of the test.

 

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 (LAUS, 2015). The following mean in vitro irritation score was calculated: 0.02. It is concluded that the test substance is not irritating to the eye.

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted according to an appropriate guideline and in compliance with GLP.
The corrosion study was a prerequisite for the performance of this irritation study.

Justification for selection of eye irritation endpoint:
The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP. There was an uncritical deviation from the guideline.

Justification for classification or non-classification

Based on the available in vitro data, α-methylenebenzyl acetate does not meet the criteria for classification for skin or eye irritation according to Regulation (EC) No. 1272/2008.