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Toxicological information

Skin irritation / corrosion

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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:
19 May, 2015 - 29 June, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2012)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): IN-1000
- Appearance: Dark amber liquid
- Storage condition of test material: In refrigerator (2-8°C) container flushed with nitrogen

Test animals

Species:
human
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.3 - 37.2
- Humidity (%): 73 - 91

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 25 μL (no correction for the purity)

NEGATIVE CONTOL:
- Amount applied: 25 µL Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µL
- Concentration: 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 15-EKIN-025). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

TREATMENT
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty five μl of the undiluted test substance was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. In addition three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: tissue viability
Value:
106
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 7%. (migrated information)

In vivo

Irritant / corrosive response data:
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with IN-1000 compared to the negative control tissues was 106%. Since the mean relative tissue viability for IN-1000 was above 50% IN-1000 is considered to be non-irritant.

Any other information on results incl. tables

Test for reduction of MTT by the test substance:

IN-1000 was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 508547). Because solutions did turn blue / purple and/or a blue / purple precipitate was observed it was concluded that IN-1000 did interfere with the MTT endpoint.

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 IN-1000 was 3.3% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues.

Acceptability of the assay:

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (0.576 -1.352).

The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
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 IN-1000 is not irritating in the in vitro skin irritation test.

Executive summary:
In an in vitro skin irritation test using a human skin model ( EPISKIN Small Model) conducted according to OECD 439 guideline and GLP principles, the influence of IN-1000 on the viability of human skin was tested. The test substance (25 μL) was applied directly to 0.38 cm^2 cultured skin. Reliable positive and negative controls were included. After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 7% whereas the test substance showed cell viability of 106%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that IN-1000 is not irritating in the in vitro skin irritation test.