(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

Endpoint summary

Description of key information

Skin irritation (in vitro, 15 minutes) – Irritating

Skin corrosion (in vitro, 3 minutes and 1 hour) – Non-corrosive

Eye irritation (in vitro, 10 minutes) – Non-irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Apr 2017 - 02 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS

Version / remarks:

published 2003, last (6th) revision 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch: 09892701
- Purity: 99.9%
- Appearance: Colourless 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 animal used as source of test system:

N/A

Justification for test system used:

Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential .

The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison with untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS). The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.

Vehicle:

unchanged (no vehicle)

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).

Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, 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 EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.

EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 30 May 2017.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL

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.

Irritation / corrosion parameter:

other: Relative absorbance

Run / experiment:

15 Minute exposure

Value:

7.1

Negative controls validity:

valid

Remarks:

Set to 100%

Positive controls validity:

valid

Remarks:

5.3

Remarks on result:

positive indication of irritation

Results after treatment with MEDOL-10 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	0.098	0.096	0.097	0.059	0.062	6.7	6.4	7.1
	0.102	0.096	0.099	0.061		6.9
	0.102	0.107	0.105	0.067		7.6

* 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 7.1% (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: 6.4 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:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the experimental conditions reported, MEDOL-10 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 MEDOL-10 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 7.1%. 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, MEDOL-10 is irritant to skin according to UN GHS and EU CLP regulation.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Apr 2017 - 11 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”

Version / remarks:

07 November 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: MEDOL-10
- Batch: 09892701
- Purity: 99.9%
- Appearance: Colourless 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 animal used as source of test system:

N/A

Justification for test system used:

Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives (Optional sub-category 1A, optional sub-category 1B and 1C) and non-corrosives. The test protocol may also enable the distinction between severe and less severe skin corrosives.

The In vitro Skin Corrosion: Human Skin Model Test is based on the observation that skin corrosion (necrotic damage of viable skin cells) shows a high correlation with skin cell cytotoxicity, occurring rapidly after brief exposure of the skin barrier (stratum corneum) to a corrosive chemical. It is designed to predict and classify the skin corrosivity potential of a chemical by using a three-dimensional human epidermis model. The epidermis model (e.g. EpiDermTM) is derived from human keratinocytes and consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK, which are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation at the cutting edge of in vitro skin technology. Ultrastructurally, the skin models closely parallel human skin. The In vitro Skin Corrosion: Human Skin Model Test consists of topical application of the test material to the tissue for 3 minutes and 1 hour, followed by immediate determination of the cytotoxic effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the exposure period.

Vehicle:

unchanged (no vehicle)

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

MTT-Solution
The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).

Cell Culture
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 ∅).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 μL

NEGATIVE CONTROL
- Amount(s) applied: 50 μL

POSITIVE CONTROL
- Amount(s) applied: 50 μL

TEST ITEM PREPARATION
50 µL (79.4 µL/cm2 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:

100.9

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:

81.1

Negative controls validity:

valid

Remarks:

Set to 100%

Positive controls validity:

valid

Remarks:

4.3%

Remarks on result:

no indication of irritation

Results after treatment with MEDOL-10 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

Conclusions:

Under the reported experimental conditions, MEDOL-10 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 MEDOL-10 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 (100.9%) after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 81.1%. 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 MEDOL-10 is non corrosive to skin according to EU CLP and UN GHS.

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:

12 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

26 July 2013

Deviations:

no

Principles of method if other than guideline:

Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch: 09892701
- Purity: 99.9%
- Appearance: Colourless liquid
- Expiry Date: 06 March 2018
- Storage Conditions: At room temperature, protected from light

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

Irritation parameter:

in vitro irritation score

Value:

> 0.63 - < 2.9

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	Proposed in vitro Irritancy 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
MEDOL-10	3.00*		-0.007*		2.90	2.93	Not categorized
	3.00*		-0.029*		2.57
	1.00*		-0.025*		0.63

* corrected values

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions reported, MEDOL-10 is not categorized (GHS).

Executive summary:

his in vitro study was performed to assess the corneal damage potential of MEDOL-10 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 MEDOL-10 did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 2.03. According to OECD 437 (see table in chapter 3.8.3) the test item is not categorized (GHS).

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The in vitro study was performed (Envigo CRS GmbH, 2017, Study DY06CT) to assess the corrosive potential of MEDOL-10 by means of the Human Skin Model Test with EpiDerm™ tissues models. The study was performed according to the EU Method B.40, the OECD test guideline 431, and MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT), and in compliance with GLP.

 

MEDOL-10 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. 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.

 

3-minute exposure to MEDOL-10 did not decrease (100.9%) the relative absorbance value, whereas 1 hour of exposure did reduce the relative absorbance value to 81.1%. 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.

 

Under the experimental conditions of this study, MEDOL-10 was found to be non corrosive to skin.

 

 

The in vitro study was performed (Envigo CRS GmbH, 2017, Study TN59TF) to assess the irritation potential of MEDOL-10 by means of the Human Skin Model Test. The study was performed according to the EC Method B.46, the OECD TG 439, and in compliance with GLP.

 

MEDOL-10 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. 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.

 

Three tissues of the human skin model EpiSkin™ were treated with MEDOL-10, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes. After treatment with MEDOL-10 the mean relative absorbance value decreased to 7.1%. This value is below the threshold for irritancy of ≤ 50%.

 

Under the experimental conditions reported, MEDOL-10 is irritant to skin according to EU CLP regulation.

 

 

The Bovine Corneal Opacity and Permeability (BCOP) test (Envigo CRS GmbH, 2017, Study CX78WH) was used to identify if MEDOL-10 could induce serious eye damage (UN GHS Category 1), or if it does not require classification for eye irritation or serious eye damage (UN GHS No Category) using the corneas of eyes isolated from cattle. The test was conducted according to the OEDC test guideline 437, and in compliance with GLP.

 

The undiluted MEDOL-10 was applied for 10 minutes followed by an incubation period of 120 minutes at 32 ± 1 °C. Negative and positive control items were tested concurrently. The decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

 

Relative to the negative control, MEDOL-10 did not cause a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 2.03. According to OECD 437 BZA does not require classification under the GHS.

Justification for classification or non-classification

Based on the above studies, MEDOL-10 meets the criteria for classification as a skin irritant. According to Commission Regulation 1272/2008, MEDOL-10 is classified as Skin Irritant, Category 2; the related Signal word is "Warning", and the Hazard Statement is "H315: Causes skin irritation".

Based on the above studies, MEDOL-10 does not meet the criteria for classification as corrosive to skin does not require classification under the CLP.

As noted above, in the OECD 437 study MEDOL-10 did not cause a relevant increase of the corneal opacity or permeability, therefore does not require classification under the CLP.