Registration Dossier

Diss Factsheets

Administrative data

Description of key information

In Vitro Skin Corrosivity

Non corrosive to the skin.

In Vitro Skin Irritation

Non-irritant to skin.

In Vitro Eye Irritation

Non-irritant.

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

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 April 2017 to 13 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
OECD Guidelines for the Testing of Chemicals, No. 431, (29 July 2016) “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test
Version / remarks:
Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further details specified in the study report.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Details on animal used as source of test system:
EPISKIN TM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-015, Expiry Date: 17 April 2017) is a three-dimensional human epidermis model. Adult human derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
Justification for test system used:
The EPISKIN TM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
INDICATOR FOR POTENTIAL FALSE VIABILITY
Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.

Check-method for possible direct MTT reduction with test item
20 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours and then any colour change was observed:
-Test items which do not react with MTT: yellow
-Test items reacting with MTT: blue or purple
After three hours of incubation, yellow colour of the mixture was detected; therefore additional controls were not used in the experiment.

Check-method to detect the colouring potential of test item
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol (simulating a tissue humid environment). As the test item had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step:
MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

PERFORMANCE OF THE STUDY
Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere.

Application (Day 0)
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test item was applied evenly to the epidermal surface of each of two test units and each additional control skin units and then 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (23.9-24.6°C) covered with the plate lids.

Rinsing (Day 0)
After the incubation time (4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT test (Day 0)
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (±15 minutes), protected from light.

Formazan extraction (Day 0)
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Cell viability measurements (Day 1)
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
20 mg of test item was applied evenly to the epidermal surface of each of two test units and each additional control skin units and then 100 μL physiological saline was
added to the test item to ensure good contact with the epidermis.
Duration of treatment / exposure:
Single exposure
Duration of post-treatment incubation (if applicable):
The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (23.9-24.6°C) covered with the plate lids.
Number of replicates:
Two epidermis units were used for each test or control materials.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
87.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ADDITIONAL CONTROLS
As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.003, Non Specific Colour% (NSCliving%) was calculated as 0.3% (see Table 1). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.
As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2. The mean OD value for the test item treated skin samples showed a 87.4% relative viability.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (1.070).
The two positive control treated tissues showed 0.5% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 20.2%.
The difference of viability between the two negative control tissue samples in the MTT assay was 1.3%.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues

Additional control

Optical Density (OD)

NSC% (living)

 

Measured

Blank corrected

Treated with

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

1

0.049

0.003

0.3

2

0.049

0.003

Mean

--

0.003

Notes:

1. Mean blank value was 0.046

2. Optical density means the mean value of the supplicate wells for each sample (rounded to three decimal places).

 

Optical Density (OD) and the calculated relative viability % of the samples

Substance

Optical Density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative Control:

Physiological saline

(0.9% (w/v) NaCl)

1

1.123

1.077

100.7

2

1.109

1.063

99.3

Mean

--

1.070

100.0

Positive Control:

Glacial acetic acid

1

0.053

0.007

0.6

2

0.050

0.004

0.3

Mean

--

0.005

0.5

Test Item:

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

1

1.076

1.030

96.3

2

0.887

0.841

78.6

Mean

--

0.935

87.4

Notes:

1. Mean blank value was 0.046

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places)

HISTORICAL CONTROL DATA

 

Negative control (Physiological saline)

Positive control

(Glacial acetic acid)

Minimum optical density (OD)

0.611

0.005

Maximum optical density (OD)

1.516

0.051

Mean optical density (OD)

0.871

0.017

Standard Deviation (SD)

0.164

0.010

Number of cases

81

81

Note: All optical density (OD) values measured are background corrected values (measured at 570 ± 30 nm)

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in this in vitro EPISKIN™(SM) model test with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch number: C034J0059), the results indicate that the test item is non corrosive to the skin, UN GHS Classification: No Category.
Executive summary:

An in vitro skin corrosivity test of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

 

Disks of EPISKINTM(SM) (two units) were treated with 6,6’-di-tert-butyl-2,2’-thiodip-cresol test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

 

Following exposure with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the mean cell viability was 87.4% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

 

In conclusion, in this in vitro EPISKIN™(SM) model test with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch number: C034J0059), the results indicate that the test item is non corrosive to the skin, UN GHS Classification: No Category.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 April 2017 to 07 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
OECD Guidelines No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (28 July 2015)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No 761/2009 of 23 July 2009, ANNEX III, B.46., “In Vitro Skin Irritation Reconstructed Human Epidermis Model Test”, amended by Commission Regulation (EU) No 640/2012 of 6 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further information specified in the study report.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Source strain:
not specified
Details on animal used as source of test system:
Human Skin
EPISKINTM (SM) (Manufacturer: SkinEthic, France, Batch No.:17-EKIN-014, Expiry Date: 10 April 2017) is a three-dimensional human epidermis model. Adult human derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Quality Control
EPISKINTM (SM) kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.
The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EPISKINTM (SM) test kits used in the present study).

Storage
The EPISKINTM (SM) kit was kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.
Justification for test system used:
The EPISKINTM (SM) model has been validated for irritation testing in an international validation study [10] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
INDICATOR FOR POTENTIAL FALSE VIABILITY
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.

Check-method for possible direct MTT reduction with test item
Approximately 10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: yellow
-Test items reacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected in the test tube.
Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

Check-method to detect the colouring potential of test-items
Prior to treatment, the test item was evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). As the test items had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test items to stain the epidermis by using additional control tissues.

Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

Application and rinsing (Day 0)
Test Item
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 10 mg of the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
Negative and positive controls
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.8-25.9°C).
After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The test item stuck on surface of the epidermis additional rinsing was used. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

MTT test (Day 2)
After the 42 hours incubation, all EPISKINTM (SM) units (except the two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere.

Formazan extraction (Day 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (Day 2)
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
10 mg of the test item was applied evenly to the epidermal surface.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.8-25.9°C).
Duration of post-treatment incubation (if applicable):
42 hours incubation
Number of replicates:
In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test items were coloured, two additional test item-treated tissues were used for the non-specific OD evaluation.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
76.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the three negative control tissues was in the recommended range (0.654). Standard deviation of the viability results for negative control samples was 1.7.
The positive control treated tissues showed 5.1% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.3.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.5.
The mean OD value of the blank samples (acidified isopropanol) was 0.048.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

ADDITIONAL CONTROLS

As no colour change (yellow colour) was observed after three hours of incubation of the test items in MTT working solution, thus the test materials did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.

As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.009, Non Specific Colour % was calculated as 1.4%. This value was below 5%, therefore additional data calculation was not necessary.

 

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissue

Additional control

Optical Density (OD)

NSC%

 

Measured

Blank corrected

Treated with

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

1

0.058

0.009

1.4

2

0.058

0.009

Mean

--

0.009

Notes:

1. Mean blank value was 0.048

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

VIABILITY RESULTS

The OD values for the test item treated skin samples showed 76.5% relative viability.

 

Optical Density (OD) and the calculated relative viability % of the samples

Substance

Optical Density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative Control:

Phosphate buffered saline

1

0.690

0.642

98.2

2

0.704

0.656

100.3

3

0.712

0.663

101.5

Mean

--

0.654

100.0

Positive Control:

5% (w/v) SDS solution

1

0.086

0.037

5.7

2

0.072

0.023

3.6

3

0.088

0.039

6.0

Mean

--

0.033

5.1

Test Item:

6,6’-di-tert-butyl-2,2’-thiodi-p-cresol

1

0.529

0.480

73.5

2

0.583

0.534

81.7

3

0.535

0.486

74.4

Mean

--

0.500

76.5

Notes:

1. Mean blank value was 0.048

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

 

HISTORICAL CONTROL DATA

 

Negative control (PBS)

Positive control

(5% (w/v) SDS solution)

Mean optical density (OD)

0.802

0.094

Standard deviation

0.157

0.048

Minimum optical density (OD)

0.573

0.032

Maximum optical density (OD)

1.362

0.354

Number of cases

111

102

PBS: Phosphate buffered saline

SDS: Sodium dodecyl sulphate

OD: Optical density (absorbance)

Note: All OD values (measured at 570 ± 30 nm) are background corrected values.

Interpretation of results:
GHS criteria not met
Conclusions:
In the in vitro EPISKIN model test with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch number: C034J0059), the results indicate that the test item is non-irritant to skin.
Executive summary:

An in vitro skin irritation test of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

 

Disks of EPISKINTM(SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

 

Following exposure with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the mean cell viability was 76.5% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

 

In conclusion, in this in vitro EPISKIN model test with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch number: C034J0059), the results indicate that the test item is non-irritant to skin, UN GHS Classification: No Category.

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:
04 April 2017 to 20 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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:
OECD Guidelines for the Testing of Chemicals 438 (26th July 2013)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
EU Commission Regulation (EC) No 1152/2010 (8th December 2010) amending, Regulation (EC) No 440/2008: Method B 48.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
OPPTS 870.2400 (EPA 712-C-98-195) August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further details specified in the study report.
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
CHICKEN HEADS COLLECTION AND TRANSPORT
Strain of chicken: ROSS 308
Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within 2 hours of collection.

SELECTION AND PREPARATION OF EYES FOR THE TEST
Eyes selection
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluoresceintreated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

Identification
The eyes were identified by chamber number, marked on the door of the chamber.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
In each experiment, 30 mg of the test item was applied onto the entire surface of the cornea
Duration of treatment / exposure:
exposure period of 10 seconds from the end of the application the cornea surface
Duration of post- treatment incubation (in vitro):
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Number of animals or in vitro replicates:
One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole in each experiment.
Details on study design:
THE BASELINE ASSESSMENTS
The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

TEST PROCEDURE
Treatment
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In each experiment, 30 mg of the test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
In each experiment negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered Imidazole.
One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole in each experiment.

Test item removal
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.
Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed. The test item treated eyes were rinsed additional gentle rinsing with 20 mL saline after treatment in each experiment.

OBSERVATION
Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
Irritation parameter:
cornea opacity score
Remarks:
mean maximum
Run / experiment:
Experiment I
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Remarks:
mean
Run / experiment:
Experiment I
Value:
0.17
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Remarks:
mean maximum
Run / experiment:
Experiment II
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Remarks:
mean
Run / experiment:
Experiment II
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test item 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results.
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

TEST ITEM

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0%

I

Mean maximum corneal swelling at up to 240 min

0.0%

I

Mean maximum corneal opacity

0.50

I

Mean fluorescein retention

0.17

I

Other observations

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) was cleared at 30 minutes after the post-treatment rinse.

Overall OCE Class

3xI

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0%

I

Mean maximum corneal swelling at up to 240 min

0.6%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other observations

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) was cleared at 30 minutes after the post-treatment rinse.

Overall OCE Class

3xI

 

POSITIVE CONTROL

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

9.7%

II

Mean maximum corneal swelling at up to 240 min

28.1%

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other observations

Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse

Overall OCE Class

1xIII 2xIV

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

9.7%

II

Mean maximum corneal swelling at up to 240 min

25.8%

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other observations

Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Overall OCE Class

1xIII 2xIV

 

NEGATIVE CONTROL

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0%

I

Mean maximum corneal swelling at up to 240 min

0.0%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other observations

None

Overall OCE Class

3xI

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0%

I

Mean maximum corneal swelling at up to 240 min

0.0%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other observations

None

Overall OCE Class

3xI

 

VALIDITY OF THE TEST

Historical Control data

 

Negative Control: Physiological Saline

Observation

Min. Value

Max. Value

Maximum corneal swelling at up to 75 min

-3.2%

3.4%

Maximum corneal swelling at up to 240 min

-4.8%

3.4%

Maximum corneal opacity change

0.00

0.50

Fluorescein retention

0.00

0.50

Number of studies

254

 

Positive Control: Imidazole

Observation

Min. Value

Max. Value

Maximum corneal swelling at up to 75 min

-6.6%

25.0%

Maximum corneal swelling at up to 240 min

-15.9

36.7%

Maximum corneal opacity change

3.50

4.00

Fluorescein retention

2.00

3.00

Number of studies

117

 

 

SUMMARY TABLE FOR UN GHS CLASSIFICATION

EXPERIMENT I AND EXPERIMENT II

Criteria for No Category

True/False

3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint class as II:

True

Test item was not stuck to the cornea:

True

Criteria for Category 1

True/False

2 or more endpoints classed as IV:

False

Corneal opacity3 at 30 min (in at least 2 eyes):

False

Corneal opacity = 4 at any time point (in at least 2 eyes):

False

Severe loosening of epithelium (in at least 1 eye):

False

Criteria for No predication can be made

True/False

Based on the endpoints not classifiable for No Category and for Category 1:

False

Particles of test item were stuck to the cornea and could not be washed off during the study

False

 

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the in vitro eye irritation assays in isolated chicken eyes with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the test item was non-irritant, UN GHS Classification: No Category.
Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (26 July 2013).

 

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiment was considered to be valid.

 

Experiment I: No corneal swelling was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5 on all three eyes) was observed on three eyes. No significant fluorescein retention change (severity 0.5 on one eye) was noted on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

 

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four hour observation period on test item treated eyes. No cornea opacity change and no fluorescein retention change were observed on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

 

Based on these in vitro eye irritation assays in isolated chicken eyes with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the test item was non-irritant, UN GHS Classification: No Category.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 August 2017 to 20 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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: Crl:KBL(NZW) New Zealand White rabbit
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
Hygienic level at arrival: SPF
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: Approx. 10 weeks old
Sex: Female (nulliparous and non-pregnant)
Body weight range
on the day of treatment: 2219 g – 2251 g
before euthanasia: 2279 g – 2304 g
Date of receipt: 10 August 2017
Acclimatisation time: 6-7 days
Animal identification: The animals were identified by indelible ink on the ear. The cages were marked with individual identity cards with information about study code, sex, cage number, dose and individual animal number.

HUSBANDRY
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
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.
Number of animal room: 034
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.2 – 22.3 °C
Relative humidity: 39 – 63 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity values were measured continuously. The measured range was checked at least daily 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: 0004181216, expiry date: 06 September 2017

WATER SUPPLY AND QUALITY CONTROL
The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system.
The drinking water is routinely analysed and is 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 three 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:
An amount of 0.1 g of the test item was administered to the animals.
Duration of treatment / exposure:
As the solid test item remained in the eye sac in all animals at the one-hour observation time point, the treated eye of test animals was rinsed with physiological saline solution.
Observation period (in vivo):
The eyes were examined 1, 24, 48 and 72 hours after treatment.
Number of animals or in vitro replicates:
Initially only one rabbit was treated with test item. The local effects were non-irritant (all scores were zero) at 24 hours, then two further rabbits were treated with the test item.
Details on study design:
TESTING PROCEDURE
In vitro study results
An in vitro eye irritation assay (CiToxLAB study code: 17/080-038CS) in isolated chicken eyes with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch number: C034J0059) concluded that the test item is non-irritant.
As the test item cannot be identified as causing serious damage or as a product beyond the limits of classification for eye irritation and severe eye damage, it is concluded that an in vivo study is required for classification. The Sponsor confirmed this statement.

Identification of pH
The pH of the test item was measured as pH 5.5, permitting the test item to be used in the animal studies.

Pre-study examination
Three female 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.

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.
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 (SC) under direct Veterinary supervision. The systemic opiate analgesic was again injected ~12 hours after the post-treatment analgesic, then every 12 hours, and NSAID injected every ~24 hours.
Systemic opiate analgesic: Bupaq (buprenorphine) 0.01 mg/kg.
Topical ocular anaesthetic: Benoxi (oxybuprocaine) one-two drops per eye.
Nonsteroidal anti-inflammatory drug: Loxicom (meloxicam) 0.5 mg/kg.

ADMINISTRATION OF THE TEST ITEM
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 test item was supplied as a fine dust therefore it was applied as supplied.
The untreated contralateral eye served as the control.

OBSERVATIONS AND SCORING
Clinical Observations and Evaluation of Ocular Irritation
The eyes were examined 1, 24, 48 and 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 ketamine 10% (Ketanest) and xylazine 2% (Nerfasin) followed by i.v. pentobarbital sodium (Release). 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.

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).

Measurement of Body Weight
Individual body weight was recorded on the day of treatment and at the end of observation period.
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
Max. score:
3
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
Max. score:
3
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
Max. score:
3
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 (IPR) or any Pain Reaction (PR) was observed during the experimental period.
Animal 1 (No: F23695) clinical observation
At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 3) were noted in the rabbit. Test item remained in the eye sac.
At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.
Animal 2 (No: F23205) clinical observation
At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 2) were noted in the rabbit. Test item remained in the eye sac.
At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.
Animal 3 (No: F23403) clinical observation
At one hour after the application, conjunctival redness (score 2), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Test item remained in the eye sac.
At 24, 48 and 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 the 72-hour observation of the second and third animals.
During the study, the control eye of each animal was symptom-free.
The general state and behaviour of animals were normal throughout the study period.
Other effects:
MORTALITY
There was no mortality observed during the study.

BODY WEIGHTS
The body weight of the animals was considered to be within the normal range of variability.

CLINICAL OBSERVATION
General daily examination: There were no clinical signs observed that could be related to treatment.

INDIVIDUAL SCORES FOR OCULAR IRRITATION

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.: F23695

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

2

3

0

0

0

0

0

*

24 h

0

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

* Test item remnant was seen in the eye sac at one hour.

 

Animal No.: F23205

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

2

2

0

0

0

0

0

*

24 h

0

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

* Test item remnant was seen in the eye sac at one hour.

 

Animal No.: F23403

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

0

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

* Test item remnant was seen in the eye sac at one hour.

 

MEAN VALUES OF EYE IRRITATION

(24, 48, 72 hour readings)

Animal Number

Sex

Cornea Opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

F23695

Female

0.00

0.00

0.00

0.00

0.00

F23205

Female

0.00

0.00

0.00

0.00

0.00

F23403

Female

0.00

0.00

0.00

0.00

0.00

 

BODY WEIGHT DATA

Animal Number

Before treatment

(g)

Before euthanasia

(g)

Body weight gain

(g)

F23695

2231

2302

71

F23205

2219

2279

60

F23403

2251

2304

53

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.
The study result triggers the following classification/labelling:
- Regulation (EC) No 1272/2008 (CLP): none
- GHS (rev. 6) 2015: none
Executive summary:

An acute eye irritation study of the test item, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 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 test item was administered as a single dose.

 

The eyes were examined at 1, 24, 48, 72 hours after application in three animals.

 

No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.

 

Animal 1 (No: F23695) clinical observation

At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 3) were noted in the rabbit. Test item remained in the eye sac.

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

Animal 2 (No: F23205) clinical observation

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

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

 

Animal 3 (No: F23403) clinical observation

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

At 24, 48 and 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 the 72-hour observation of the second and third animals.

 

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        0.00               0.00               0.00

Cornea          0.00               0.00               0.00

Iris                 0.00               0.00               0.00

 

The test item, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.

The study result triggers the following classification/labelling:

- Regulation (EC) No 1272/2008 (CLP): none

- GHS (rev. 6) 2015: none

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In Vitro Skin Corrosivity

An in vitro skin corrosivity test of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol test item was performed in a reconstructed human epidermis model. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

 

Disks of EPISKINTM(SM) (two units) were treated with 6,6’-di-tert-butyl-2,2’-thiodip-cresol test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

 

Following exposure with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the mean cell viability was 87.4% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In Vitro Skin Irritation

An in vitro skin irritation test of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

 

Disks of EPISKINTM(SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

 

Following exposure with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the mean cell viability was 76.5% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In Vitro Eye Irritation

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (26 July 2013).

 

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

 

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiment was considered to be valid.

 

Experiment I: No corneal swelling was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5 on all three eyes) was observed on three eyes. No significant fluorescein retention change (severity 0.5 on one eye) was noted on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four hour observation period on test item treated eyes. No cornea opacity change and no fluorescein retention change were observed on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

 

Based on these in vitro eye irritation assays in isolated chicken eyes with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the test item was non-irritant.

In Vivo Eye Irritation

An acute eye irritation study of the test item, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol 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 test item was administered as a single dose.

The eyes were examined at 1, 24, 48, 72 hours after application in three animals.

 

No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.

Animal 1 (No: F23695) clinical observation

At one hour after the application, conjunctival redness (score 2), chemosis (score 2) and discharge (score 3) were noted in the rabbit. Test item remained in the eye sac.

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

Animal 2 (No: F23205) clinical observation

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

At 24, 48 and 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.

Animal 3 (No: F23403) clinical observation

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

At 24, 48 and 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 the 72-hour observation of the second and third animals.

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, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.

Justification for classification or non-classification

In Vitro Skin Corrosivity

In this in vitro EPISKIN™(SM) model test with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch number: C034J0059), the results indicate that the test item is non corrosive to the skin, UN GHS Classification: No Category.

In Vitro Skin Irritation

In this in vitro EPISKIN model test with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch number: C034J0059), the results indicate that the test item is non-irritant to skin, UN GHS Classification: No Category.

In Vitro Eye Irritation

Based on the in vitro eye irritation assays in isolated chicken eyes with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the test item was non-irritant, UN GHS Classification: No Category.

In Vivo Eye Irritation

The test item, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 24 hours.

The study result triggers the following classification/labelling:

- Regulation (EC) No 1272/2008 (CLP): none

- GHS (rev. 6) 2015: none