Registration Dossier

Administrative data

Description of key information

In Vitro Skin Irritation: Not irritating using an in vitro human skin model test.

In Vitro Skin Corrosion: Not corrosive using an in vitro human skin model test.

In Vitro Eye Irritation: Not irritating in the Bovine Corneal Opacity and Permeability Test (PCOP test).

In Vivo Eye Irritation: Not irritating to the eyes of rabbits.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 July 2015 to 18 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Cell type:
non-transformed keratinocytes
Cell source:
other: cultured
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
Test system
EpiDerm Skin Model (EPI-200, Lot no.: 22299 kit O).
The model 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.

Rationale
Recommended test system in international guidelines (OECD and EC).

Source
MatTek Corporation, Ashland MA, U.S.A.

Cell culture
Tissues
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 (Dulbecco’s Modified Eagle’s Medium)medium.

DMEM
Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) medium
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions
All incubations, with the exception of the test substance 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 66 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 - 36.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30.5 to 35.1 mg of the solid test substance.
Duration of treatment / exposure:
3 minutes & 1 hour
Duration of post-treatment incubation (if applicable):
Not specified
Number of replicates:
2 tissues per test group (3 minutes exposure, 1 hour exposure, negative control, positive control).
Details on study design:
Test substance preparation
The correction factor is not applicable for the test, therefore no correction was made for the purity of the test item.
The solid test substance (30.5 to 35.1 mg) was crushed and ground in a mortar with pestle to improve the consistency and was applied directly on top of the skin tissue.

Reference substances
Negative control: Milli-Q water (Millipore Corp., Bedford, Mass., USA).
Positive control: Potassium hydroxide (KOH; Merck, Darmstadt, Germany), an 8.0 normal solution was prepared.

Study design
Test for the interference of the test substance with the MTT endpoint
A test substance may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test substance is present on the tissues when the MTT viability test is performed.

Test for colour interference by the test substance
The test substance was checked for possible colour interference before the study was started. Some non-coloured test substances may change into coloured substances in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, approximately 25 mg of the test substance 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.
In case the test substance induces colour interference in aqueous conditions, in addition to the normal procedure, two tissues must be treated with test substance for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues will be incubated with DMEM medium.

Test for reduction of MTT by the test substance
2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, approximately 25 mg of the test substance 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 was observed.
In case the test substance reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues must be used for the cytotoxicity evaluation with MTT.

Application/Treatment of the test substance
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 1 hour at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test substance was applied. The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] and two for a 1-hour exposure. The skin was moistened with 25 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and 30.5 to 35.1 mg of the solid test substance 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. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

Cell viability measurement
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 substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme(s):
REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature and humidity.
Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
91
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
87
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritant / corrosive response data:
Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 91% and 87%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive.
Other effects:
The test substance was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test substance did not interfere with the MTT endpoint.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 7%.

The maximum inter-tissue variability in viability between two tissues treated identically was less than 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 10%. It was therefore concluded that the test system functioned properly.

Mean absorption in the in vitro skin corrosion test with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodeylthio)propionate]

 

3-minutes application

1-hour application

A (OD570)

B (OD570)

Mean (OD570)

 

SD

A (OD570)

B (OD570)

Mean (OD570)

 

SD

Negative control

2.041

1.991

2.016

±

0.035

2.208

2.026

2.117

±

0.128

Test substance

1.799

1.855

1.827

±

0.039

1.769

1.895

1.832

±

0.089

Positive control

0.152

0.132

0.142

±

0.014

0.197

0.237

0.217

±

0.028

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background adsorption (0.0425). Isopropanol was used to measure the background adsorption.

 

Mean tissue viability in the in vitro skin corrosion test with 2,2-Bis[[3-(dodecylhio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

 

3-minute application viability (percentage of control)

1-hour application viability (percentage of control)

Negative control

100

100

Test substance

91

87

Positive control

7

10

 

INDVIDUAL OD MEASUREMENTS AT 570 NM

 

3-minute application (OD570)

1-hour application (OD570)

A

B

A

B

Negative control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

2.105

2.084

2.061

 

2.052

2.035

2.014

 

2.274

2.247

2.229

 

2.095

2.043

2.067

Test substance

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

1.872

1.822

1.830

 

1.900

1.903

1.889

 

1.834

1.810

1.790

 

1.954

1.939

1.918

Positive control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

0.193

0.195

0.197

 

0.175

0.176

0.174

 

0.238

0.244

0.237

 

0.282

0.277

0.281

OD = Optical density

Duplicate exposure are indicated by A and B.

 

HISTORICAL CONTROL DATA FOR IN VITRO SKIN CORROSION STUDIES

 

Negative control

Positive control

Positive control

3-minute treatment (OD570)

1-hour treatment (OD570)

3-minute treatment (OD570)

1-hour treatment (OD570)

3-minutes treatment (% viability)

1-hour treatment (% viability)

Range

1.076 – 2.167

1.361 – 2.203

0.017 – 0.29

0.063 – 0.226

6 – 16

4 – 12

Mean

1.78

1.78

0.15

0.12

10.6

7.0

SD

0.25

0.20

0.05

0.04

2.9

2.1

N

43

47

44

42

22

22

SD = Standard deviation

N = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period April 2012 to April 2015.

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
2,2-Bis[[3-(dodecylthio)-1-xopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] Naugard 412S is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

In vitro skin corrosion test with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], Naugard 412S, using a human skin model.

This report describes the ability of the test substance to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200). The possible corrosive potential of the test substance was tested through topical application for 3 minutes and 1 hour.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method (adopted 26 September 2014).

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

 

Batch T3J07001 of the test substance consisted of white to off white pellets with a purity of 97.8%. Skin tissue was moistened with 25 μl of Milli-Q water and approximately 25 mg of the test substance was applied directly on top of the skin tissue.

 

The positive control had a mean relative tissue viability of 7% after 3 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 maximum inter-tissue variability in viability between two tissues treated identically was less than 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 10%, indicating that the test system functioned properly.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 91% and 87%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive.

 

Finally, it is concluded that this test is valid and that 2,2-Bis[[3-(dodecylthio)-1-xopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], Naugard 412S, is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 July 2015 to 07 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Bovine eyes
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: 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 but within 4 hours.
Transport: 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:
323.8 to 375.2 mg.
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
90 minutes
Duration of post- treatment incubation (in vitro):
Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C
Number of animals or in vitro replicates:
3 corneas per test group (test substance, negative control, positive control).
Details on study design:
Test substance preparation
Since a correction factor was not applicable for this test, no correction was made for the purity/composition of the test substance.
Since no workable suspension of the test substance in physiological saline could be obtained, the test substance was used as delivered by the sponsor and added pure on top of the corneas. Before application the test substance was crushed and ground in a mortar with pestle to improve the consistency.

Reference substances
Negative control: A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
Positive control: 20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

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 (Eagle’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 MC2 (Clermont-Ferrand, France) 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.

Cornea selection and Opacity reading
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 (OP-KIT, MC2, Clermont-Ferrand, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (323.8 to 375.2 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. 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 positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance 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 (Merck) 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 5 mg
Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). 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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.
Irritation parameter:
in vitro irritation score
Run / experiment:
Single exposure, 240 minutes of treatment
Value:
0.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity

Mean Permeability

Mean In vitro Irritation Score1,2

Negative control

0.0

0.000

0.0

Positive control

113.3

2.729

154.3

Test substance

-0.7

0.001

-0.7

1Calculated using the negative control mean opacity and mean permeability values.

2In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490value).

 

INDIVIDUAL OPACITY, PERMEABILITY AND IN VITRO SCORES

 

Opacity score

Treatment

Opacity before treatment

Opacity after treatment

Final Opacity1

Negative control corrected Final Opacity2

Mean Opacity

 

Negative control

2

3

1

0.0

0.0

1

3

2

1.0

2

2

0

-1.0

 

Mean final opacity: 1.0

 

 

Positive control

1

99

98

97.0

113.3

1

115

114

113.0

4

135

131

130.0

 

Test substance

2

2

0

-1.0

-0.7

1

1

0

-1.0

3

4

1

0.00

1Final Opacity = Opacity after treatment – Opacity before treatment

2Negative control correct Final Opacity = Final opacity – Mean final opacity negative control

 

Permeability score individual values (corrected)

Treatment

Dilution factor

OD4901

OD4902

OD4903

Average OD

Final OD

Mean final negative control

 

Negative control

1

0.032

0.032

0.036

0.033

0.033

0.022

1

0.015

0.016

0.018

0.016

0.016

1

0.015

0.016

0.018

0.016

0.016

 

Positive control

6

0.392

0.390

0.392

0.391

2.348

 

6

0.351

0.351

0.350

0.351

2.104

6

0.691

0.680

0.695

0.689

4.132

 

Test substance

1

0.008

0.005

0.005

0.006

0.006

1

0.038

0.041

0.039

0.039

0.039

1

0.023

0.023

0.024

0.023

0.023

 

Permeability score individual values (corrected)

Treatment

Dilution factor

Negative control corrected OD49011

Negative control corrected OD49021

Negative control corrected OD49031

Negative control corrected OD490Average

Negative control corrected final OD490

Average OD

 

Negative control

1

0.010

0.010

0.014

0.011

0.011

0.000

1

-0.007

-0.006

-0.004

-0.006

-0.006

1

-0.007

-0.006

-0.004

-0.006

-0.006

 

Positive control

6

0.370

0.368

0.370

0.369

2.216

2.729

6

0.329

0.329

0.328

0.329

1.972

6

0.669

0.658

0.673

0.667

4.000

 

Test substance

1

-0.014

-0.017

-0.017

-0.016

-0.016

0.001

1

0.016

0.019

0.017

0.017

0.017

1

0.001

0.001

0.002

0.001

0.001

1OD490values corrected for the mean final negative control permeability (0.022).

 

In Vitro irritancy score

Treatment

Negative control corrected Final Opacity

Negative control corrected Final OD490

In vitro Irritancy Score1

 

Negative control

0.0

0.011

0.2

1.0

-0.006

0.9

-1.0

-0.006

-1.1

 

Positive control

97.0

2.216

130.2

113.0

1.972

142.6

130.0

4.000

190.0

 

Test substance

-1.0

-0.016

-1.2

-1.0

0.017

-0.7

0.0

0.001

0.0

1In vitro irritancy score (IVIS) = opacity value + (15 x OD490value).

 

HISTORICAL CONTROL DATA

Historical control data for the BCOP studies

 

Negative control

Positive control

Opacity

Permeability

In vitro Irritancy Score

In vitro Irritancy Score

Range

-3 – 2

-0.056 – 0.050

-3.3 – 2.1

68 – 135

Mean

-0.22

0.00

-0.28

95.22

SD

0.94

0.01

0.95

18.75

N

94

96

93

73

SD = Standard deviation

N = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of April 2012 to April 2015.

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.7 after 240 minutes of treatment.
Executive summary:

Evaluation of the eye hazard potential of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] using the Bovine Corneal Opacity and Permeability test (BCOP test).

The report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test substance was tested through topical application for approximately 240 minutes.

 

The study procedures described in the report were based on the most recent OECD and EC guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 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”(adopted July 26, 2013).

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants ". Official Journal of the European Union No. L324; Amended by EC No. 1152/2010 No. L142, 09 December 2010.

 

Batch T3J07001 of the test substance consisted of a white to off white pellet with a purity of 97.8%. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (323.8 to 375.2 mg).

 

The negative control responses for opacity and permeability with an IVIS ranging from -1.1 to 0.9 were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 154 and above the historical positive control current mean plus two standard deviations. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.7 after 240 minutes of treatment.

 

Since 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 May 2017 to 25 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
The relative humidity (min. 28.90 and max. 72.41 %) was out of the target range (30-70 %) during the study. This deviation is considered to have no impact on the outcome of the study and interpretation of the results.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
No further details specified in the study report.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Species and strain: New Zealand White rabbits
Source: S&K-LAP Kft., 2173 Kartal, Császár út 135, Hungary
Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies.
Animal health: Only animals in acceptable health condition were used for the test. Both eyes of each animal provisionally selected for testing were examined prior to starting the study. Animals showing eye irritation, ocular defects or pre-existing corneal injury were not used.
Number of animals: 3 animals
Age of animals at treatment: 12-13 weeks old (young adult)
Sex: Male
Body weight range
on the day of treatment: 3410 g – 3772 g
before euthanasia: 3513 g – 3886 g
Date of receipt: 27 April 2017
Acclimatization time: at least 19 days
Animal identification: The individual identification was by engraved ear tag. The cages were marked with individual identity cards with information about study code, sex, dose, cage number and individual animal number.

HUSBANDRY
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Number of animal room: 032
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature during the study: 17.58 – 22.27 °C
Relative humidity during the study: 28.90 – 72.41%
Housing/Enrichment: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity values were measured continuously. The measured range was checked during the acclimatisation and experimental phases.

FOOD AND FEEDING
Animals received UNI diet for rabbits produced by Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út 0399, Hungary, ad libitum. Animals were provided with the following batch:
0003992970, expiry date: 20 June 2017

WATER SUPPLY AND QUALITY CONTROL OF WATER
The animal received tap water, fit for human consumption, ad libitum, from the automatic system supplied by the communal water network. The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The quality control analysis is performed once every 3 months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 g
Duration of treatment / exposure:
Single treatment / 1 hour
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
Initially only one rabbit was treated with test item. The local effect in the first rabbit shows scores of above zero but not severe, then a second rabbit was treated after the 48-hour observation of the first rabbit. Then as the result in the second rabbit was not severe, a third rabbit was treated after the 96-hour observation of the second rabbit.
Details on study design:
TESTING PROCEDURE
Identification of pH
The pH was assessed to identify if it was extreme before application to animals. The pH of the test item was accessed by measurement of the supernatant from a 1% w/v mixture in distilled water according to CIPAK MT75. The pH was found to be 6.0, so the test item is permitted for use in animal studies.

Pre-study examination
Three male animals in acceptable health condition were selected for the test. Care was taken to select only those animals that had a normal eye condition and any with ocular lesions were rejected.

Chronology of animal use
Initially only one rabbit was treated with test item. The local effect in the first rabbit shows scores of above zero but not severe, then a second rabbit was treated after the 48-hour observation of the first rabbit. Then as the result in the second rabbit was not severe, a third rabbit was treated after the 96-hour observation of the second rabbit.

Analgesic and anaesthetic treatment
Sixty minutes (60 ±10 min) prior to test substance application, a systemic opiate analgesic was administered by subcutaneous injection (SC) under direct Veterinary supervision. Repeat injections were given on the first day as appropriate to maintain an adequate level of analgesia.
Five minutes (5 ±1.5 min) prior to test substance application, a topical ocular anaesthetic was applied to each eye (including the control eye to ensure direct comparison of any ocular observations).
Eight hours (8 to 9 hr) after test substance application, a systemic opiate analgesic and a nonsteroidal anti-inflammatory drug (NSAID) were administered by subcutaneous injection under direct Veterinary supervision. The systemic opiate analgesic was again injected ~12 hours after the post-treatment analgesic and then every 12 hours, and NSAID injected every ~24 hours, until eye scores were zero.
Systemic opiate analgesic: Bupaq (0.3 mg/mL Buprenorphine) 0.01 mg/kg.
Topical ocular anaesthetic: Benoxi (4 mg/mL Oxybuprocaine chloride) one-two drops/eye.
Nonsteroidal anti-inflammatory drug: Loxicom (5 mg/mL Meloxicam) 0.5 mg/kg.

ADMINISTRATION OF THE TEST ITEM
Dosage
A single amount of 0.1 g of the solid test item was powdered and administered as a single dose.

Application of the Test Item
The test substance was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for at least one second in order to prevent loss of the material.
The untreated contralateral eye served as the control.

Duration of Exposure
The treated eye of test animals was rinsed with physiological saline solution, as the irritation scores more than one and test item remained in the eye sac at one hour after application.

OBSERVATIONS AND SCORING
Clinical Observations and Evaluation of Ocular Irritation
The eyes were examined at 1, 24, 48, 72 hours after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill-health during the study were recorded. At the end of the observation period, the animal was sacrificed by intramuscular injections of Ketanest (100 mg/mL ketamine) and Nerfasin (20 mg/mL xylazine) followed by i.v. pentobarbital sodium. Death was verified by checking pupil and corneal reflex and the absence of respiration.
All rabbits were examined for distress at least twice daily, with observations at least 6 hours apart. Clinical observations or signs of ill-health were recorded.

Measurement of Body Weight
Individual body weight was recorded on the day of treatment and at the end of observation period of each animal.

Scoring and Assessment of Local Reaction
The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (02 October 2012).
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
Examination of eye-irritancy
No Initial Pain Reaction/Pain reaction (IPR/PR) was observed.

Animal 1 (No: 924) clinical observation
At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Solid test item remained in the eye sac.
At 24 hours after the application, conjunctival redness (score 2) was noted in the rabbit.
At 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

Animal 2 (No: 901) clinical observation
At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Solid test item remained in the eye sac.
At 24 hours after the application, conjunctival redness (score 1) was noted in the rabbit.
At 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

Animal 3 (No: 914) clinical observation
At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Solid test item remained in the eye sac.
At 24 and 48 hours after the application, conjunctival redness (score 1) was noted in the rabbit.
At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.
Other effects:
There were no clinical signs observed that could be related to treatment.
As no clinical signs were observed, the experiment was terminated after 72 hours observation of the third rabbit. During the experiment, the control eye of each animal was symptom-free.
The general state and behaviour of animals were normal throughout the study period.

INDIVIDUAL SCORES FOR OCULAR IRRITATION

 

Study Code: 17/040-005N                    Species: NZW Rabbit

Dose: 0.1 g                                         Sex: Male

Start of Exposure: 16/18/22 May 2017         Test Item: 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

 

Abbreviations:

R = Redness               OD = Opacity degree of density

CH = Chemosis           OE = Extent of opaque area

D = Discharge             IPR/PR = Initial or any pain reaction

0 = Normal (in case of control eye and other lesions)

 

Animal No.: 924

Time

Score of irritation

IPR/PR

Other sign

Conjunctivae

Cornea

Iris

Control eye

R

CH

D

OD

OE

R

Pre-treatment

0

0

0

0

0

0

0

0

0

Post-treatment

(h = hour)

1 h

2

1

1

0

0

0

0

0

*

24 h

2

0

0

0

0

0

0

0

0

48 h

0

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

0

* solid test item remained in the eye sac.

 

Animal No.: 901

Time

Score of irritation

IPR/PR

Other sign

Conjunctivae

Cornea

Iris

Control eye

R

CH

D

OD

OE

R

Pre-treatment

0

0

0

0

0

0

0

0

0

Post-treatment

(h = hour)

1 h

2

1

1

0

0

0

0

0

*

24 h

1

0

0

0

0

0

0

0

0

48 h

0

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

0

* solid test item remained in the eye sac.

 

Animal No.: 914

Time

Score of irritation

IPR/PR

Other sign

Conjunctivae

Cornea

Iris

Control eye

R

CH

D

OD

OE

R

Pre-treatment

0

0

0

0

0

0

0

0

0

Post-treatment

(h = hour)

1 h

2

1

1

0

0

0

0

0

*

24 h

1

0

0

0

0

0

0

0

0

48 h

1

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

0

* solid test item remained in the eye sac.

 

MEAN VALUES OF EYE IRRITATION

(24, 48, 72 hour reading)

Animal Number

Sex

Cornea Opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

924

Male

0.00

0.00

0.67

0.00

0.00

901

Male

0.00

0.00

0.33

0.00

0.00

914

Male

0.00

0.00

0.67

0.00

0.00

 

BODY WEIGHT DATA

Animal Number

Before treatment

(g)

Before euthanasia

(g)

Body weight gain

(g)

924

3410

3513

103

901

3520

3602

82

914

3772

3886

114

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], applied to rabbit eye mucosa, caused conjunctival effects at one and 24 hours after the treatment which were fully reversible within 72 hours. There were no corneal effects observed in the study.
According to Regulation (EC) No 1272/2008, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] does not require classification as an eye irritant.
According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] does not require classification as an eye irritant.
Executive summary:

An acute eye irritation study of the test item 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2012). Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification.

 

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 g of the solid test item was powdered and administered as a single dose.

 

The eyes were examined at 1, 24, 48 and 72 hours after application.

 

No Initial Pain Reaction/Pain reaction (IPR/PR) was observed.

 

Animal 1 (No: 924) clinical observation

At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Solid test item remained in the eye sac.

At 24 hours after the application, conjunctival redness (score 2) was noted in the rabbit.

At 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

Animal 2 (No: 901) clinical observation

At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Solid test item remained in the eye sac.

At 24 hours after the application, conjunctival redness (score 1) was noted in the rabbit.

At 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

Animal 3 (No: 914) clinical observation

At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Solid test item remained in the eye sac.

At 24 and 48 hours after the application, conjunctival redness (score 1) was noted in the rabbit.

At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

As no clinical signs were observed, the experiment was terminated after 72 hours observation of the third rabbit. During the experiment, the control eye of each animal was symptom-free.

 

The general state and behaviour of animals were normal throughout the study period.

 

No mortality occurred during the study. The bodyweights of all rabbits were considered to be within the normal range of variability.

 

The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

Animal 1        Animal 2        Animal 3

Chemosis      0.00               0.00               0.00

Discharge      0.00               0.00               0.00

Redness         0.67               0.33               0.67

Cornea           0.00               0.00               0.00

Iris                   0.00               0.00               0.00

 

The test item 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], applied to rabbit eye mucosa, caused conjunctival effects at one and 24 hours after the treatment which were fully reversible within 72 hours. There were no corneal effects observed in the study.

 

According to Regulation (EC) No 1272/2008, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] does not require classification as an eye irritant.

 

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3- (dodecylthio)propionate] does not require classification as an eye irritant.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In Vitro Skin Irritation

In vitro skin irritation test with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] using a human skin model.

The report describes the ability of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes. 

Skin tissue was moistened with 5 μl of Milli-Q water and 12.7 to 18.5 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. 

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 115%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 13% after 15 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 less than 10%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

 

In Vitro Skin Corrosion

In vitro skin corrosion test with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], Naugard 412S, using a human skin model.

The report describes the ability of the test substance to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200). The possible corrosive potential of the test substance was tested through topical application for 3 minutes and 1 hour.

Skin tissue was moistened with 25 μl of Milli-Q water and approximately 25 mg of the test substance was applied directly on top of the skin tissue.

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 91% and 87%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive. 

Finally, it is concluded that this test is valid and that 2,2-Bis[[3-(dodecylthio)-1-xopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], Naugard 412S, is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

 

In Vitro Eye Irritation

Evaluation of the eye hazard potential of 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] using the Bovine Corneal Opacity and Permeability test (BCOP test).

The report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of the test substance was tested through topical application for approximately 240 minutes.

Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas (323.8 to 375.2 mg).  

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.7 after 240 minutes of treatment.

 

In Vivo Eye Irritation

An acute eye irritation study of the test item 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2012). Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification.

 

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A single amount of 0.1 g of the solid test item was powdered and administered as a single dose.

The eyes were examined at 1, 24, 48 and 72 hours after application.

 

No Initial Pain Reaction/Pain reaction (IPR/PR) was observed.

 

Animal 1 (No: 924) clinical observation

At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Solid test item remained in the eye sac.

At 24 hours after the application, conjunctival redness (score 2) was noted in the rabbit.

At 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

Animal 2 (No: 901) clinical observation

At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Solid test item remained in the eye sac.

At 24 hours after the application, conjunctival redness (score 1) was noted in the rabbit.

At 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

Animal 3 (No: 914) clinical observation

At 1 hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Solid test item remained in the eye sac.

At 24 and 48 hours after the application, conjunctival redness (score 1) was noted in the rabbit.

At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

As no clinical signs were observed, the experiment was terminated after 72 hours observation of the third rabbit. During the experiment, the control eye of each animal was symptom-free.

 

The general state and behaviour of animals were normal throughout the study period.

No mortality occurred during the study. The bodyweights of all rabbits were considered to be within the normal range of variability.

 

The test item 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], applied to rabbit eye mucosa, caused conjunctival effects at one and 24 hours after the treatment which were fully reversible within 72 hours. There were no corneal effects observed in the study.

 

Justification for selection of skin irritation / corrosion endpoint:

Endpoint derived in GLP laboratory test in accordance with OECD Guideline 431 and EU Method B.40.

 

Justification for selection of eye irritation endpoint:

Endpoint conclusion derived in GLP laboratory test in accordance with OECD Guideline 437 and EU Method B.47.

Justification for classification or non-classification

Skin Irritation

2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] is non-irritant in the in vitro skin irritation test under the experimental conditions, no classification is required for skin irritation.

Skin Corrosion

2,2-Bis[[3-(dodecylthio)-1 -oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], is not corrosive in the in vitro skin corrosion test under the experimental conditions, no classification is required for skin corrosion.

Eye Irritation

Since 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

According to Regulation (EC) No 1272/2008, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] does not require classification as an eye irritant.

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3- (dodecylthio)propionate] does not require classification as an eye irritant.