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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1-3 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: An in vitro study, according to OECD test guidelines and GLP compliant, based on the EPISKIN model has been completed. The data are considered reliable for completing this endpoint

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Principles of method if other than guideline:
In vitro skin irritation test based on reconstructed human epidermis model
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
dilithium(1+) disodium bis(2-methylpropanoate) bis(3-hydroxy-2,2,4-trimethylpentanoate)
EC Number:
939-200-6
IUPAC Name:
dilithium(1+) disodium bis(2-methylpropanoate) bis(3-hydroxy-2,2,4-trimethylpentanoate)
Constituent 2
Reference substance name:
Sodium Isobutyrate Solution
IUPAC Name:
Sodium Isobutyrate Solution
Test material form:
other: reaction mass semi-liquid solution
Details on test material:
- Name of test material (as cited in study report): Sodium Isobutyrate Solution

- Substance type:Reaction mass
- Physical state: Light yellow semi liquid
- Analytical purity: 86.5%

- Composition of test material, percentage of components: confidential information

- Purity test date: 12 April 2013
- Lot/batch No.: W195_02
- Expiration date of the lot/batch: 17 October 2013

- Storage condition of test material:room temperature

Test animals

Species:
other: iin vitro - reconstructed human epidermis model
Strain:
other: not applicable
Details on test animals or test system and environmental conditions:
Test model - EPISKIN reconstructed human epidermis model. EPISKIN-SM is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. 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. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a percentage. The % reduction in viability is used to predict the irritation potential of the material under investigation. The in vitro assay is an accepted alternative to the conduct of a rabbit skin irritation assay.

IN-LIFE DATES: From: 1 May 2013 To: 3 May 2013

Test system

Type of coverage:
other: in vitro assay
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
20 mg of the semi-liquid test material were applied to each epidermal surface in the test wells.
50 µL of phosphate buffered saline negative control applied to three control skin units
50 µL of 5% SDS applied to three positive control skin units
Duration of treatment / exposure:
On Day -1 maintenance medium was pre-warmed to 37°C and applied to the assay plate wells (2 mL per well). The epidermal constructs were overlaid on the medium and the prepared wells were incubated overnight at 37°C.

On day 0, 20 mg of test material was applied to the epidermal surface of three prepared wells. 50 µL of PBS or SDS applied to each of three wells to act as negative or positive controls. The plates were exposed for 15 minutes at circa 25°Cand then the membranes were removed and rinsed in PBS to remove test material residues. The epidermal surface was then vacuum-cleaned. The epidermal unit was replaced onto fresh medium and incubated for 42 hours at 37°C.
After 42 hours the epidermal units were transferred to wells filled with MTT solution and the units incubated or a further 3 hours at 37°C.
Formazan extraction occurred at the end of the MTT incubation. A disk of epidermis obtained by biopsy punch was separated into the epidermal and collagen matrix fractions and each was placed into 500 µL of acidified isopropanol. Formazan extraction proceeded during a two-hour incubation. Following extraction the absorbance/optical density (OD) was recorded for each sample using spectrophotometry at 540 nm.
Observation period:
42 hours
Number of animals:
Not applicable. Three replicate wells prepared for test material and three each for the positive and negative controls
Details on study design:
Test model - EPISKIN reconstructed human epidermis model. EPISKIN-SM is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. 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. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a percentage. The % reduction in viability is used to predict the irritation potential of the material under investigation. The in vitro assay is an accepted alternative to the conduct of a rabbit skin irritation assay.

EPISKIN-SM (Source: SkinEthic, France, Batch No.: 13-EKIN-016, Expiry date: 06 May 2013) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeIts 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 viabilityded 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 a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

The negative control use was phosphate buffered saline (PBS). The positive control was 5% sodium dodecyl sulphate in distilled water.

Control checks were included fordetermination of false viability, for possible direct MTT reduction by the test material and for any colouring potential arising directly from coloured test materials that mayhave affected the OD from the spectrophotometry fluid or by direct staining of the tissues..

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: mean OD as an indicator of cell viability
Value:
98
Remarks on result:
other:
Remarks:
Basis: other: percent relative viability. Time point: 42 hours. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Following exposure to Sodium Isobutyrate Solution, the mean relative viability value of the treated skins was 98% ; the substance was not irritating. All validity criteria were within acceptable limits and therefore the study can be considered as valid. (migrated information)

In vivo

Irritant / corrosive response data:
Following exposure to Sodium Isobutyrate Solution, the mean relative viability value of the treated skins was 98%. THe test substance was not therefore irritating to skin. All validity criteria were within acceptable limits and therefore the study can be considered as valid

Any other information on results incl. tables

The results of the optical density (OD) measured at 540 nm of each extract and the calculated % viability of the cells is presented in Table 1:

 

Table 1: Optical Density (OD) and the calculated % viability of the cells

 

Substance

Optical Density (OD)

Viability (% RV)

Negative Control:

1

0.680

102

PBS

2

0.630

95

 

3

0.687

103

 

mean

0.666

100

 

Standard deviation

4.36

Positive Control:

1

0.047

7.1

5%SDS

2

0.052

7.8

 

3

0.038

5.7

 

mean

0.046

6.9

 

Standard deviation

1.07

Test Item:

1

0.692

104

Sodium Isobutyrate Solution

2

0.517

78

 

3

0.737

111

 

mean

0.649

98

 

Standard deviation

17.39

 

The OD value for the test item treated skin was a viability of 98%.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
Following exposure to Sodium Isobutyrate Solution, the mean relative viability value of the treated skins was 98%; the test substance was not therefore irritating to skin
Executive summary:

The reconstructed human epidermis model EPISKIN-SM is designed to predict and classify the skin irritant potential of chemicals, by measuring its cytotoxic effect, as reflected in the MTT (Thiazolyl blue) cell viability assay, on the EPISKIN reconstituted human epidermis.

Disks of EPISKIN (three units / chemical) were treated with sodium isobutyrate solution and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. 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 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritating to skin.

Following exposure to Sodium Isobutyrate Solution, the mean relative viability of the treated skin was 98% and therefore the substance was not considered to be irritating to skin. All validity criteria were within acceptable limits and therefore the study can be considered as valid. Based on this study, Sodium Isobutyrate Solution was not found to be irritating to the skin and does not meet the criteria for classification for skin irritation.

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