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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:
Between 09June 2009and 15 June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: See Principles of method in the following section
Deviations:
not applicable
Principles of method if other than guideline:
The principle of the assay is based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-la in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N'-bis[3-(dimethylamino)propyl]-9-nonyl-10-octylnonadecanediamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-[(2E)-oct-2-en-1-yl]-4-pentylcyclohexyl]nonanamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-octyl-4-pentylcyclohexyl]nonanamide
EC Number:
937-688-5
Cas Number:
1391530-05-4
Molecular formula:
C46H82N4O2 to C46H94N4O2
IUPAC Name:
N,N'-bis[3-(dimethylamino)propyl]-9-nonyl-10-octylnonadecanediamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-[(2E)-oct-2-en-1-yl]-4-pentylcyclohexyl]nonanamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-octyl-4-pentylcyclohexyl]nonanamide

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST TISSUE: EPISKIN reconstituted human epidermis model
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37 °C

Test system

Type of coverage:
other: in vitro testing
Preparation of test site:
other: in vitro testing
Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control and Sodium Dodecyl Sulphate (SDS) was used as the positive control (this is an in vitro test)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ul test material was added to 2 ml culture media
VEHICLE: none
Duration of treatment / exposure:
15 min
Observation period:
42 h incubation after treatment
Number of animals:
Triplicate
Details on study design:
TEST Tissue was cultured in a 12 well plate.
REMOVAL OF TEST SUBSTANCE
- Washing (if done): 15 min after application
SCORING SYSTEM: MTT assay

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: The relative mean viability
Value:
0.988
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Reversibility: other: not applicable. Remarks: This is an in vitro study. (migrated information)

In vivo

Other effects:
None

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test material turned blue which indicated that the test material directly reduced MTT and therefore the MTT viability assay was performed in parallel on viable and freeze-killed tissues.

Test Material, Positive Control Material and Negative Control Material

The direct reduction of MTT by the test material was less than 30% of the negative control value. Therefore, the net OD of the test material killed tissue was subtracted from the mean OD of the test material treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

The relative mean viability of the test material treated tissues, following correction for the direct reduction of MTT, was 98.8% after a 15-minute exposure.

Following the 15-minute exposure the test material treated tissues appeared blue which was considered indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤ 40% relative to the negative control treated tissues and the Standard Deviation (SD) value of the % viability was ≤ 20%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was ≥ 0.6 and the SD value of the % viability was ≤ 20%. The negative control acceptance criterion was therefore satisfied.

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be Non-Irritant.
Executive summary:

Introduction.The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN~~reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-la in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the

non-irritant result.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was found to have the ability to directly reduce MTT. Therefore it was necessary to apply the test material to a single water-killed tissue to allow for the differentiation of true and false viability. At the end of the exposure period each tissue was rinsed before incubating for approximately 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. 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 u1 samples were transferred to the appropriate wells of a pre-labelled

96-well plate. The optical density was measured at 540 nm.

Data are presented in the form of % viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: The relative mean viability of the test material treated tissues was 98.8% after a 15-minute exposure.

Quality criteria:The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:The test material was considered to be Non-Irritant.