Di-tert-dodecyl disulphide

Endpoint summary

Description of key information

In vitro skin irritation

The skin irritation potential of DI-TERT-DODECYL DISULPHIDE was evaluated in the EpiskinTM reconstructed human epidermis model (Richez, 2016a). The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46. The study was conducted in compliance with the principles of Good Laboratory Practice. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 100% with a standard deviation of 9%. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.

In vitro eye irritation

The potential irritant and corrosive properties of DI-TERT-DODECYL DISULPHIDE to the eye was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test (Richez, 2016b). This method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The design of this study was based on the guideline OECD Guideline 437 and the study was performed in compliance with the OECD Principles of Good Laboratory Practice. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C. A single experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item was applied undiluted in a single experiment. The test item, negative and positive controls were evaluated using a treatment time of 10 minutes and using the open-chamber method. At the completion of the treatment period, all formulations were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on the three corneas treated with the test item. All acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0. As the mean IVIS was=3, DI-TERT-DODECYL DISULPHIDE was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 November 2015 -- 14 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 439 (In vitro dermal irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EpiskinTM Model Kit (0.38 cm2 tissues)

Cell source:

other: SkinEthic Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: several
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: no data
- Wavelength: 570 nm
- Filter: no data
- Filter bandwidth: no data

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
SCORING SYSTEM:
- Optical density (OD) was measured at 570 nm:
Relative mean viability (%) = 100 x mean cOD(test item) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank
- mean cOD Positive Control = mean ODPC - mean ODblank
DECISION CRITERIA
Mean relative viability is = 50%: category 2 (H315)
Mean relative viability is > 50% and Mean IL-1a released concentration > 60 pg/mL: category 2 (H315)
Mean relative viability is > 50% and Mean IL-1a released concentration = 60 pg/mL: no category

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

Amount/concentration applied
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ìL
VEHICLE
- Amount(s) applied (volume or weight with unit): 10ìL
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10ìL
- Concentration (if solution): 5% (w/v) aqueous solution

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transform
ation by viable cells

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Time point: 15 min exposure + 42h expression

Value:

100

Vehicle controls validity:

valid

Remarks:

100%

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

9%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.

Executive summary:

The skin irritation potential of DI-TERT-DODECYL DISULPHIDE was evaluated in the EpiskinTM reconstructed human epidermis model. The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46. The study was conducted in compliance with standard operating procedures and the principles of Good Laboratory Practice. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 100% with a standard deviation of 9%. As the mean viability was > 50% after the MTT reduction, the results met the criteria for a non-irritant response.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2015 -- 10 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 437 (In vitro eye irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine cornea

Details on test animals or tissues and environmental conditions:

Species: bovine cattle.
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
Age: bovine cattle were up to 12 months old.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from Supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.

Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used within a maximum of 24 hours.
Storage of the corneas: as the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

(Pre)Incubation T°C: 32°C
Dates of experimental phase: from 01 December 2015 to 10 December 2015

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

Exposure period of 10 minutes (± 30 seconds) followed by rinsing

Duration of post- treatment incubation (in vitro):

Opacity measurement:
- before treatment
- after 2-hour incubation in water bath at +32°C (± 1°C).

Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement

Number of animals or in vitro replicates:

Triplicate corneas for each timepoint and tested substance (test item, negative control, positive control)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Rinsing: the anterior part of the eye was emptied and then rinsed 7 times with pre-warmed cMEM

NEGATIVE CONTROL:
As the test item was tested undiluted (i.e. in its original form), the following negative control was used:
- Name: 0.9% Sodium Chloride (NaCl)
- Batch No.: A444
- Supplier: Lavoisier

Since several test items were assayed concurrently, the negative control was shared.

POSITIVE CONTROL:
As the test item is a non-surfactant containing mixture (tested using a 10-minute treatment), the positive control was 10% sodium hydroxide solution (10% NaOH).
The positive control was prepared by CiToxLAB France pharmacy on the morning of use by dissolving anhydrous pellets of sodium hydroxide (batch No. SZBE3360V supplied by Sigma-Aldrich) in water obtained by reverse osmosis using ELIX 5 (Millipore SA).

Since several test items were assayed concurrently, the positive control was shared.

SCORING SYSTEM/TOOL
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Interpretation: see below

Irritation parameter:

in vitro irritation score

Run / experiment:

10 minutes

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

214

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

10 minutes

Value:

-0.3

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

130

Irritation parameter:

fluorescein leakage

Value:

0.004

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

5.626

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
No notable opaque spots or irregularities were observed on the three test item and negative control-treated corneas.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item was identified as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

The potential irritant and corrosive properties of DI-TERT-DODECYL DISULPHIDE to the eye was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test. This method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The design of this study was based on the guideline OECD Guideline 437 and the study was performed in compliance with with the OECD Principles of Good Laboratory Practice. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C. A single experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item was applied undiluted in a single experiment. The test item, negative and positive controls were evaluated using a treatment time of 10 minutes and using the open-chamber method. At the completion of the treatment period, all formulations were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on the three corneas treated with the test item. All acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0. As the mean IVIS was =3, DI-TERT-DODECYL DISULPHIDE was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

DI-TERT-DODECYL DISULPHIDEt is not classified for skin and eye irritation according to CLP and GHS Regulations.