Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

Endpoint summary

Description of key information

Skin :

- Skin irritation (OECD 439, K, Rel.1): Irritating to skin.

- Skin corrosion (OECD 431, K, Rel.1): not classified for skin corrosion.

<=> The test substance can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. 

 

Eye:

- Eye corrosion (OECD 437, BCOP, K, Rel.1): mean IVIS = 1.5 (IVIS ≤ 3). The test item is predicted not corrosive/not severely irritating to the eye (No category) with the Bovine Corneal Opacity and Permeability test method.

<=> The test substance is not classified for the eye irritation/corrosion hazard. No hazard statement or signal word “Warning” is required. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 to 09 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.431 and under GLP compliance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Dated to 29 July 2016.

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

other: reconstituted epidermis (EpiDerm Skin Model)

Cell type:

other: EpiDerm Skin Model

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
- The EpiDerm Skin Model (EPI-200, Lot no.: 24940 kits J and K) consists of normal, human-derived epidermal keratinocytes 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Source: MatTek Corporation, Ashland MA, U.S.A.
- Rationale: Recommended test system in international guidelines (OECD and EC).

CELL CULTURE
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium (Supplemented DMEM medium, serum-free supplied by MatTek Corporation). The plates were incubated for approximately 1.5 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test item was applied.

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 47 - 82%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

EVALUATION OF DIRECT INTERACTION WITH MTT
- Colour interference by the test item in aqueous conditions: To assess the colour interference, 50 μl of the test item or 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Reduction of MTT by the test item: To assess the ability of the test item to reduce MTT, 50 μl of the test item was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.
> Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.

TREATMENT
The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. 50 μl of the undiluted test item was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively.

REMOVAL OF TEST MATERIAL
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM), both
supplied by MatTek Corporation, was prepared.
The DMEM medium was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.

PREDICTION MODEL / DECISION CRITERIA:

- Acceptability of the assay:
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8)
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

In addition to the quality criteria a comparison of laboratory historical data on negative and positive controls was made to verify the functioning of the test system.

- Data evaluation and statistical procedures:
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute
treatment is considered corrosive if the relative tissue viability after 1-hour treatment with
the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the
negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below
15%.

The following information presents the data interpretation and optional sub-categorisation in case a test item will be corrosive.

Viability measured after 3-minutes and 1 hour

STEP 1 (corrosive or not corrosive)
< 50% after 3 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND < 15% after 60 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure ==> Non-corrosive

STEP 2 (subcategories for items identified as corrosive in step 1)
< 25% after 3 min exposure ==> Optional Sub-category 1A
≥ 25% after 3 min exposure ==> A combination of optional Sub-categories 1B-and-1C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

During 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.

Duration of post-treatment incubation (if applicable):

The skin sample was placed in MTT solution of 1 mg/mL concentration for 3 hours at 37°C± 1°C, 5% CO2.

Number of replicates:

2 living human skin models for each time

Irritation / corrosion parameter:

% tissue viability

Value:

95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 3 minutes

Irritation / corrosion parameter:

% tissue viability

Value:

112

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 60 minutes

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues, after 3 minutes exposure, was 95%, versus 7% in the positive control.
- The mean percent viability of the treated tissues, after 1 hour exposure, was 112 %, versus 14% in the positive control.

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	± SD	Mean viability %	Viability difference between replicates %
Negative control	A	1.7327 1.7449 1.7441	1.698	1.658	± 0.056	100.00	4.7
	B	1.6579 1.6502 1.6745	1.6118
Positive control	A	0.1701 0.1674 0.1657	0.125	0.120	± 0.008	7	9.0
	B	0.1577 0.1562 0.1553	0.114
Test item	A	1.6523 1.7420 1.6830	1.650	1.567	± 0.117	95	10
	B	1.5378 1.5326 1.5101	1.484

 

 

 

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Skin	OD	Mean OD / disc (#)	Mean OD / product	± SD	Mean viability %	Viability difference between replicates %
Negative control	A	1.4840 1.4800 1.4840	1.440	1.406	± 0.049	100.00	4.8
	B	1.4386 1.3726 1.4303	1.371
Positive control	A	0.2751 0.2888 0.2664	0.234	0.202	± 0.046	14	28
	B	0.2121 0.2125 0.2126	0.170
Test item	A	1.6631 1.6308 1.5944	1.587	1.578	± 0.013	112	1.2
	B	1.6585 1.5719 1.6022	1.568

SD: standard deviation

 

In these tables the values are corrected for background absorption (0.0424). Isopropanol was used to measure the background absorption.

 

Acceptability criteria:

- The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. - The mean relative tissue viability following the 1-hour exposure to the positive control was 14% ( < 15%).
- In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 10%, indicating that the test system functioned properly (≤ 30%).

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95% and 112% respectively. Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.

Therefore, in accordance with the CLP Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”, and based on the results from the previous EpiSkin study OECD 439 as irritant to the skin, the test item can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. 

Executive summary:

An in vitro skin corrosion test according to the OECD Guideline OECD 431 and in compliance with GLP was performed.

The test item Ethyl Safranate was applied as supplied, at the dose of 50 μL to 2 living Human skin model surfaces for each time (EpiDerm (EPI-200)) for 3 minutes and 1 hour. The application was followed by a rinse with PBS to remove residual test item. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, the remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control).

 

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item and treated with the positive control item (potassium hydroxide 8N) were respectively 95% and 112% versus 7% and 14% respectively, with the positive control.

Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.

 

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95% and 112% respectively. Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.

Therefore, in accordance with the CLP Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”, and based on the results from the previous EpiSkin study OECD 439 as irritant to the skin, the test item can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. 

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 July to 29 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 439 without any deviation.

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)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN™ Reconstructed Human Epidermis Model, which is recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model TM (EPISKIN-SM TM, 0.38 cm², Batch no.: 16-EKIN-034)
-Source: SkinEthic Laboratories, Lyon, France.
- Description: Three-dimensional human epidermis model, which consists of adult human derived
epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Preparation: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 22 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

ENVIRONMENTAL CONDITIONS
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 76 -91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.9 - 36.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

TREATMENT
The first test resulted in a standard deviation above acceptance criteria for the test item treated tissues and was therefore rejected (not reported) and a repeat test was performed. The test was performed on a total of 3 tissues per test item together with negative and positive controls. 25 μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was respread after 7 minutes contact time.

REMOVAL OF TEST MATERIAL
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

CELL VIABILITY
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed. The corrected OD for each sample or control were calculated by subtracting the value of OD(blank) mean from each reading.

OD(corrected) = OD(raw)– OD(blank mean)

The OD value representing 100% cell viability is the average OD of the negative controls.
For the EpiSkin® Irritation assay this is the average OD of the negative controls.

The % Viability for each sample and positive control is calculated as follows:
% Viability = (OD(corrected)/mean OD(negative controls)) x 100

PREDICTION MODEL / DECISION CRITERIA
- Acceptability of the assay:
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤ 50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.

In addition to the quality criteria a comparison of laboratory historical data on negative and positive controls was made to verify the functioning of the test system.

- Data evaluation and statistical procedures:
A test item is considered irritant in the skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
- The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL of the test item was applied to the epidermal surface of three tissues.
- Concentration (if solution): The test item was used as supplied.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Three tissues were treated with 25 μl PBS.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): Three tissues were treated with 25 μl 5% SDS.

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate tissues for test item, negative and positive controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

11

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes; the mean OD570 for the negative control treated tissues was 0.744. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range and the standard deviation value of the percentage viability of three tissues treated identically was 7% or less, indicating that the test system functioned properly.
- Acceptance criteria met for positive control: Yes; The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 11% relative to the negative control treated tissues and the standard deviation value of the viability was 3.4%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.0%. The test item acceptance criterion was therefore satisfied.
- Historical Control Data Comparison: All values for the control groups were within the ranges obtained by the testing laboratory by collecting all data over the period of December 2012 to December 2015.

Table 7.3.1/1: Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control	0.746	0.744	0.052	100*	7.0
	0.691
	0.794
Positive Control	0.061	0.083	0.025	11	3.4
	0.076
	0.110
Test Item	0.122	0.125	0.007	17	1.0
	0.133
	0.120

 

OD=Optical Density

SD= Standard deviation

*= The mean viability of the negative control tissues is set at 100%.

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

It is concluded that the test is valid and that ETHYL SAFRANATE is irritant in the in vitro skin irritation test under the experimental conditions described in this report as the mean percent viability of the treated tissues is 17%, versus 11% in the positive control (5% Sodium Dodecyl Sulfate). In accordance with the Regulation EC No. 1272/2008 and to the GHS, and based on the results from the previous EpiDerm study OECD 431 as non-corrosive, the test item can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. 

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model.

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite^® M200 Pro Plate Reader. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). Skin irritation is expressed as the remaining cell viability after exposure to the test item.

 

The relative mean viability of the test item treated tissues was 17% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

 

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 7% or less, indicating that the test system functioned properly.

 

It is concluded that the test is valid and that ETHYL SAFRANATE is irritant in the in vitro skin irritation test under the experimental conditions described in this report as the mean percent viability of the treated tissues is 17%, versus 11% in the positive control (5% Sodium Dodecyl Sulfate). In accordance with the Regulation EC No. 1272/2008 and to the GHS, and based on the results from the previous EpiDerm study OECD 431 as non-corrosive, the test item can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. 

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:

19 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.437 and under GLP compliance.

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:

Adopted July 26, 2013.

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

other: bovine cattle (Bos taurus)

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

10 ± 1 minutes at 32 ± 1°C

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes at 32 ± 1°C

Number of animals or in vitro replicates:

Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

OPACITY MEASUREMENT
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to: 𝑂𝑝𝑎𝑐𝑖𝑡𝑦= [((𝐼0/𝐼)−0.9894) / 0.0251], with 𝐼0 the empirically determined illuminance through a cornea holder but with windows and medium, and 𝐼 the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

APPLICATION OF SODIUM FLUORESCEIN
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

PERMEABILITY DETERMINATIONS
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

One of the negative control eyes was excluded since it was slightly translucent resulting in an opacity value which was outside the normal range.

Positive controls validity:

valid

Other effects / acceptance of results:

ETHYL SAFRANATE was tested neat.

The individual in vitro irritancy scores for the negative controls ranged from -1.3 to -0.4 (study plan deviation). The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
The individual positive control in vitro irritancy scores ranged from 53 to 73 for Ethanol. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The mean in vitro irritancy score of the positive control (Ethanol) was 62 and was within two standard deviations of the current historical positive control mean.
It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The corneas treated with ETHYL SAFRANATE showed opacity values ranging from -0.1 to 2.9 and permeability values ranging from -0.013 to 0.057. The corneas were clear after the 10 minutes of treatment with ETHYL SAFRANATE. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.1 to 2.7 after 10 minutes of treatment with ETHYL SAFRANATE.
ETHYL SAFRANATE did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.5 after 10 minutes of treatment.
Since ETHYL SAFRANATE induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Table 7.3.2/1: Summary of opacity, permeability and in vitro scores

 

Treatment	Mean opacity^1	Mean permeability^1	Mean in vitro irritation score^1,2
Negative control	-1.0	0.010	-0.8
Positive control (Ethanol)	22.3	2.636	61.8
ETHYL SAFRANATE	1.2	0.019	1.5

¹ Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.
² In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

 

Individual values for opacity, permeability and in vitro scores were available in the report attached in the section 'Attached full study report'.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item was predicted and, ETHYL SAFRANATE induced an IVIS ≤ 3. Therefore, the test item is not corrosive/not severely irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on bovine corneas.

 

Corneas obtained from young slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for the minimum of 1 hour at 32 ± 1°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item was applied undiluted, in a single experiment using a treatment time of 10 minutes onto the epithelium of the cornea. Negative and positive controls were applied using the same treatment time. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 120 ± 10 minutes at 32 ± 1°C before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes) at 32 ± 1°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities. Opacity, residual test item on corneas and fluorescein fixation were observed on each cornea treated with the test item.

 

The individual in vitro irritancy scores for the negative controls ranged from -1.3 to -0.4 (study plan deviation). The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
The individual positive control in vitro irritancy scores ranged from 53 to 73 for Ethanol. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The mean in vitro irritancy score of the positive control (Ethanol) was 62 and was within two standard deviations of the current historical positive control mean.
It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The corneas treated with ETHYL SAFRANATE showed opacity values ranging from -0.1 to 2.9 and permeability values ranging from -0.013 to 0.057. The corneas were clear after the 10 minutes of treatment with ETHYL SAFRANATE. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.1 to 2.7 after 10 minutes of treatment with ETHYL SAFRANATE. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.5 after 10 minutes of treatment.
Since ETHYL SAFRANATE induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

 

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 1.5. Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item was predicted and, ETHYL SAFRANATE induced an IVIS ≤ 3. Therefore, the test item is not corrosive/not severely irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

- One key study was identified (EpiSkin, CRL, 2016).

In the in vitro skin irritation study performed according to the OECD Guideline 439 and in compliance with GLP, the EPISKIN^TM reconstructed human epidermis model was used. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite^® M200 Pro Plate Reader. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). Skin irritation is expressed as the remaining cell viability after exposure to the test item.

The relative mean viability of the test item treated tissues was 17% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The positive control had a mean cell viability of 11% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 7% or less, indicating that the test system functioned properly.

It is concluded that the test is valid and that ETHYL SAFRANATE is irritant in the in vitro skin irritation test under the experimental conditions described in this report as the mean percent viability of the treated tissues is 17%, versus 11% in the positive control (5% Sodium Dodecyl Sulfate). In accordance with the Regulation EC No. 1272/2008 and to the GHS, and based on the results from the previous EpiDerm study OECD 431 as non-corrosive, the test item can be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. 

 

Skin corrosion:

- One key study was identified (EpiDerm, CRL, 2017).

An in vitro skin corrosion test according to the OECD Guideline OECD 431 and in compliance with GLP was performed.

The test item Ethyl Safranate was applied as supplied, at the dose of 50 μL to 2 living Human skin model surfaces for each time (EpiDerm (EPI-200)) for 3 minutes and 1 hour. The application was followed by a rinse with PBS to remove residual test item. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, the remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control).

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item and treated with the positive control item (potassium hydroxide 8N) were respectively 95% and 112% versus 7% and 14% respectively, with the positive control.

Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 95% and 112% respectively. Because the mean relative tissue viability for ETHYL SAFRANATE was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered non corrosive in the in vitro skin corrosion test.

Therefore, in accordance with the CLP Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”.

 

 

 

Eye corrosion:

One key study was identified (BCOP, CRL, 2016).

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on bovine corneas.

Corneas obtained from young slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for the minimum of 1 hour at 32 ± 1°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item was applied undiluted, in a single experiment using a treatment time of 10 minutes onto the epithelium of the cornea. Negative and positive controls were applied using the same treatment time. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 120 ± 10 minutes at 32 ± 1°C before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes) at 32 ± 1°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities. Opacity, residual test item on corneas and fluorescein fixation were observed on each cornea treated with the test item.

The individual in vitro irritancy scores for the negative controls ranged from -1.3 to -0.4 (study plan deviation). The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
The individual positive control in vitro irritancy scores ranged from 53 to 73 for Ethanol. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The mean in vitro irritancy score of the positive control (Ethanol) was 62 and was within two standard deviations of the current historical positive control mean.
It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The corneas treated with ETHYL SAFRANATE showed opacity values ranging from -0.1 to 2.9 and permeability values ranging from -0.013 to 0.057. The corneas were clear after the 10 minutes of treatment with ETHYL SAFRANATE. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.1 to 2.7 after 10 minutes of treatment with ETHYL SAFRANATE. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.5 after 10 minutes of treatment.
Since ETHYL SAFRANATE induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 1.5. Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item was predicted and, ETHYL SAFRANATE induced an IVIS ≤ 3. Therefore, the test item is not corrosive/not severely irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

 

 

 

Respiratory irritation:

No data was available.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self classification:

Skin irritation/Corrosion

1/ Skin irritation (OECD 439, K, Rel.1): The mean percent viability of the treated tissues is 17%, versus 11% in the positive control (5% Sodium Dodecyl Sulfate).

2/ Skin corrosion (OECD 431, K, Rel.1): 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 95% and 112%, versus 7.0% and 14.0%, respectively, with the positive control item (potassium hydroxide 8N).

=> Based on the results from in vitro studies, the test item can be classified as skin irritant (Category 2 CLP/GHS). The hazard statement “H315: Causes skin irritation” with the signal word “Warning” is required.

 

Eye irritation/Corrosion:

1/ Eye corrosion (OECD 438, K, Rel.1): The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.5 after 10 minutes of treatment. Since ETHYL SAFRANATE induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

=> Based on these results, the test substance is not classified for the eye irritation/corrosion hazard. No hazard statement or signal word “Warning” is required. 

 

 Respiratory irritation:

No data was available regarding respiratory irritation.