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Administrative data

Description of key information

The test material is not irritating to skin but is highly irritating to the eyes.

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:
13-Feb-2012 to 23-Apr-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also complies with OECD GLP regulations.
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): S-10793
- IUPAC nomenclature - Sodium diisobutyldithiophosphinate
- Lot S-20227-170B
- Appearance - White powder with lumps
- CAS No. 13360-78-6
- Molecular Formula - C8H18PS2.Na
- Molecular Weight - 232 g/mole
- Purity 93-94%
- Expiration date of the lot/batch: 16 December 2013
- Storage condition of test material: At room temperature in the dark
Test system:
human skin model
Remarks:
EPISKIN-SM, 0.38 cm²
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin small model (EPISKIN-SM, 0.38 cm³) from SkinEthic Laboratories, Lyon, France
- Tissue batch number(s):12-EKIN-016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After 15 minutes, the tissues were washed with PBS to remove residual test substance.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3h
- Spectrophotometer: Tecan Infinite M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Living epidermis was transferred to 12 well plates and incubated with 2 ml Mili-Q for 48 +/-1 hours. After incubation, killed epidermis was stored at <= - 15 °C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.
- N. of replicates : 3
- Method of calculation used: The non-specific reduction of MTT by the test material was 21% of the negative control tisues. The net OD of the treated killed tissues was substracted from the ODs of the test substance treated viable tissues.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to the skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 44 hours of post incubation is =< 50 % of the mean viability of the negative controls.
- The test substance is considered to be non-irritating to the skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 44 hours of post incubation is > 50 % of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 17.7 to 19.2 mg, moistened with 5 µl water

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
83
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test material was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that S-10793 did interact with MTT.
Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the test material is non-irritant in the in vitro skin irritation test.

Executive summary:

In vitro skin irritation of the test material was investigated using a human skin model according to OECD TG 439 and under GLP conditions. EPISKIN-SM was used and the topical application lasted 15 minutes.

Skin tissue was moistened with 5 µl of Milli-Q water and 17.7 to 19.2 mg of the test material was applied directly on top of the skin tissue for 15 minutes. After a post-incubation period of approximately 44 hours, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. The test material did interact with 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test substance and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test material was 21% of the negative control tissues. The net optical density (OD) of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with S-10793 compared to the negative control tissues was 83%. Since the mean relative tissue viability for the test material was above 50% after 15 minutes treatment it is considered to be non-irritant.

The positive control had a mean cell viability of 4% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was 18%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that the test material is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-Feb-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also complies with OECD GLP regulations.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
September 2009
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): S-10793
- IUPAC nomenclature - Sodium diisobutyldithiophosphinate
- Lot S-20227-170B
- Appearance - White powder with lumps
- CAS No. 13360-78-6
- Molecular Formula - C8H18PS2.Na
- Molecular Weight - 232 g/mole
- Purity 93-94%
- Expiration date of the lot/batch: 16 December 2013
- Storage condition of test material: At room temperature in the dark
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Vitelco, 's Hertogenbosch, The Netherlands
- Time interval prior to initiating testing: < 24 hours
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 300 mg per cornea

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole


Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
The isolated corneas were stored at 32 °C in a petri dish with cMEM (Eagle‟s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 °C. The corneas were incubated for the minimum of 1 hour at 32 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Physiological saline solution

POSITIVE CONTROL USED
20% (w/v) Imidazole solution

APPLICATION DOSE AND EXPOSURE TIME
301 to 314 mg of the test substance applied directly on the corneo
750 µl of positive and negative controls
240 minutes at 32 °C

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times with MEM with phenol red, and then once with fresh cMEM

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post -treatment reading.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.

DECISION CRITERIA: decision criteria as indicated in the TG used
In vitro score range / Ìn vitro classification
0 - 3 / Non irritant
3.1 - 25 / Mild irritant
25.1 - 55 / Moderate irritant
≥ 55.1 / Severe irritant
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
190
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 115 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

In Vitro irritancy score

Eye

Negative control correctedFinal Opacity

Negative control correctedFinal OD490

In vitroIrritancy Score1

Negative control

1

0

0.004

0.1

2

0

-0.001

0.0

3

-1

-0.002

-1.0

Positive control

4

69

2.310

103.7

5

72

4.494

139.4

6

68

2.622

103.3

S-10793

10

116

4.818

188.3

11

117

4.278

181.2

12

122

5.280

201.2

 

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

 

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test material is a severe irritant in the Bovine Corneal Opacity and Permeability test.

Executive summary:

Screening for eye irritancy potential of the test material was investigated using the Bovine corneal opacity and permeability test (BCOP test) following OECD TG 437 and under GLP conditions. The test was done by topical application of the pure material (ca. 300 mg) for approximately 240 minutes.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score (IVIS) of the positive control (20% (w/v) Imidazole) was 115 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test material induced severe ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 190 after 240 minutes of treatment.

Since it induced an IVIS ≥ 55.1, it is concluded that the test material is a severe irritant in the Bovine Corneal Opacity and Permeability test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Additional information

The in vitro skin irritation test was conducted according to OECD 439 guidelines and GLP principles. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes of treatment with the test material compared to the negative control tissues was 83%. Since the mean relative tissue viability for S-10793 was above 50% after 15 minutes of treatment, the substance was considered a non-irritant.

A Bovine Corneal Opacity and Permeability (BCOP) test performed by NOTOX (2012) resulted in observations of an in vitro irritancy score (IVIS) of 190 following 240 minutes of exposure. This led to the conclusion that S-10793 is a "severe irritant or corrosive."


Effects on eye irritation: highly irritating

Justification for classification or non-classification

Concerning skin irritation, the substance is Not Classified based on the available data and professional judgment

Concerning eye irritation, the substance is classified as causing serious eye damage

H318 - Causes serious eye damage