Sodium methanesulphonate

Endpoint summary

Description of key information

Skin irritation

The skin irritation potential of SPS 10LS was evaluated using the Episkin^TMreconstructed 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 studywas conducted in compliance with CiToxLAB France 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 +, 5% CO₂in 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 addition, the concentration of the inflammatory mediator IL-1awas evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

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-minute exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 112% with a Standard Deviation of 2% as assessed by the MTT assay.As the mean relative viability > 50% after the MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The IL-1a concentration value of one tissue was found below the limit of quantification (< 5.00 pg/mL). Consequently, the mean IL-1a concentration from the three test item-treated tissues was not calculated. The IL-1aconcentration values of the two other test item-treated tissues were found < 60 pg/mL (i.e.6.23 and 6.65 pg/mL). Therefore, the results met the criteria for anin vitroclassification as non-irritant to skin.

Under the experimental conditions of this study, SPS 10LS is considered to be non-irritant to skin.

Eye irritation

The potential irritant and corrosive properties of SPS 10LS was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test method which 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 CiToxLAB France standard operating proceduresand 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, applied undiluted, the negative and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.The corneas were then incubated for 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 test item-treated corneas.All acceptance criteria were fulfilled. The study was therefore considered as valid.The meanIn VitroIrritancy Score (IVIS) of the test item-treated corneas was: 0.As the mean IVIS was < 3, the test item 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:

20 June 2016 - 18 July 2016

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

reconstructed human epidermis

Source species:

human

Cell type:

other: human epidermal keratinocytes

Cell source:

foreskin from multiple donors

Vehicle:

water

Details on test system:

The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis supplied by Episkin, Lyon, France.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL of the test item.
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): SDS at 5% (w/v).

Duration of treatment / exposure:

Exposure period of 15 minutes, followed by rinsing.

Duration of post-treatment incubation (if applicable):

42-hour recovery period.

Number of replicates:

Triplicate tissues for each tested substance (test item, negative control, positive control).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min exposure + 42h expression

Value:

112

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

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: mean cOD of the three negative controls should be = 0.6 and the Standard Deviation (SD) value of the % viability should be = 18%,
- Acceptance criteria met for positive control: relative mean viability of the positive control should be = 40% of the relative mean viability of the negative control and the SD value of the % viability should be = 18%.

Interpretation of results:

GHS criteria not met

Conclusions:

SPS 10LS, is considered to be non-irritant to skin.According to the results of this study, the classification of the test item should be:- no category (UN GHS and Regulation (EC) No. 1272/2008).

Executive summary:

The skin irritation potential of SPS 10LS was evaluated using the Episkin^TMreconstructed 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 studywas conducted in compliance with CiToxLAB France 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 +, 5% CO₂in 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 addition, the concentration of the inflammatory mediator IL-1awas evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

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-minute exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 112% with a Standard Deviation of 2% as assessed by the MTT assay. As the mean relative viability > 50% after the MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The IL-1a concentration value of one tissue was found below the limit of quantification (< 5.00 pg/mL). Consequently, the mean IL-1a concentration from the three test item-treated tissues was not calculated. The IL-1aconcentration values of the two other test item-treated tissues were found < 60 pg/mL (i.e. 6.23 and 6.65 pg/mL). Therefore, the results met the criteria for an in vitro classification as non-irritant to skin.

Under the experimental conditions of this study, SPS 10LS is considered to be non-irritant to skin.

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:

05 August 2016 - 08 September 2016

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

Strain:

other: not applicable

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: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 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 cooled 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°CDates of experimental phase: from 23 August 2016 to 08 September 2016

Vehicle:

unchanged (no vehicle)

Controls:

other: in vitro negative and positive controls

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

Observation period (in vivo):

Opacity measurement:- before treatment---- +- directly after exposure period----- after 2-hour incubation in waterPermeability measurement:- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement

Number of animals or in vitro replicates:

Not applicableTriplicate corneas for each 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 5 times with pre-warmed cMEM containing phenol red.NEGATIVE CONTROL:As the test item was tested undiluted (i.e. in its original form), 0.9% Sodium Chloride (0.9% NaCl) was used as negative control.Since several test items were assayed concurrently, the negative control was shared.POSITIVE CONTROL:As the test item was tested using a 10-minute treatment, the positive control was Absolute Ethanol. It was used neat and sampled on the day of use.Since several test items were assayed concurrently, the positive control was shared.SCORING SYSTEM/TOOL- Opacity:Using an opacitometerThe average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values in positive control and test item.- Permeability:Using a spectrophotometer: optical density (OD) at 490 nm wavelengthThe optical density is corrected by subtracting the average negative control value from values in positive control and test item.- Scoring:In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)Interpretation: see below

Irritation parameter:

in vitro irritation score

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

The potential irritant and corrosive properties of SPS 10LS was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test method which 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 CiToxLAB France standard operating proceduresand 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, applied undiluted, the negative and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 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 test item-treated corneas. 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, the test item 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

No classification is warranted.