Alkanol reaction products with bis(aminoalkyl(C=1~6))carbomonocycle, alkoxy(C=3~8)alkanol(C=2~7) and 2,4-TDI

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2009 --- 9 November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Name of test material (as cited in study report): 20231250
- Physical state: white powder
- Storage condition of test material: room temperature (ca. 20°C) in the dark

In vitro test system

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: 540 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 540 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 = or < 50%: category 2 (H315)
Mean relative viability is > 50% : no category

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Amount/concentration applied
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 +/- 2 mg
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution):
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution): 5% in distilled water

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 transformation by viable cells

Number of replicates:

3

Test animals

Species:

other: in vitro test

Details on test animals or test system and environmental conditions:

The test was conducted in accordance with the Standard Operating Procedure " In Vitro Skin Irritation Test: Human Epidermis Model (L’Oreal 2009)" supplied by L’Oreal (leading laboratory in the validation of the test for ECVAM) and the
OECD Guideline For The Testing of Chemicals Draft proposal for a new guideline In Vitro Skin Irritation (OECD 2008).

The test involves the application of the test substance for 15 minutes to the EPISKIN threedimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38cm2. The EPISKIN kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.
The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell death in the cell layers. The cell viability is determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Time point: 15 min exposure + 42h expression

Value:

117

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

9.6%

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

Any other information on results incl. tables

Assay validity:

The mean absorbance of the triplicate negative control values was 0.7 which was above the minimum acceptance value of 0.6. The standard deviation (SD) of the % viability was 12.0 which was below the maximum acceptance value of 18.

The mean % viability of the positive control was 9.60 ± 4.10 of the negative control. These were below the maximum acceptance values of 30% viability and SD of 18.

Table 7.3.1/1 : Episkin results

Sample	Tissue viability as percentage of mean OD negative control				Prediction
	Replicate tissues			Mean +/- SD
	a	b	c
Negative control	86.3	109	105	100 +/- 12.0	Not applicable
Positive control	8.00	6.50	14.3	9.60 +/-4.10	Irritant
Test Substance	115	113	123	117 +/- 5.40	Non irritant

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is considered to be non-irritant to skin.

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test substance using the EPISKIN^TMreconstructed human epidermis model (in vitro assay). The test procedure was equivalent to the one described in the OECD guideline 439 and the study was performed in compliance with Good Laboratory Practice..

Preliminary tests were performed to detect the ability of the test substance to directly reduce MTT as well as its colouring potential. following these preliminary tests, triplicate tissues (Episkin epidermis surface area of 0.38 cm²) were treated with 10 mg of the test item as a powder for an exposure period of 15 minutes. The tissues were wetted with 5 µL of distilled water prior to application of the test substance. At the end of the exposure period each tissue was rinsed to remove residual test substance before incubating for 42 hours at 37°C in a humidified atmosphere of 5% CO2 in air. At the end of the post-exposure incubation period each tissue was taken for MTT-loading (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm with acidified isopropanol solution as a blank.

Negative (sterile Dulbecco's Phosphate Buffered Saline (DPBS) with magnesium and calcium) and positive controls (5% sodium dodecyl sulphate (SDS) in distilled water) were used in the study. The mean absorbance of the triplicate negative control values was 0.7 which was above the minimum acceptance value of 0.6. The standard deviation (SD) of the % viability was 12.0 which was below the maximum acceptance value of 18. The mean % viability of the positive control was 9.60 +/- 4.10 of the negative control. This was below the maximum acceptance values of 30% viability and SD of 18. Therefore the study was considered as valid.

The relative mean viability of the test item treated tissues was 117 +/- 5.40 % after the 15-minute exposure period. As the mean viability was above 50% after MMTT reduction, the result met the criteria for a non-irritant response.