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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:
03 February 2009 to 09 February 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Determination of skin irritation potential using the EPISKIN (TM) Reconstituted Human Epidermis Model
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 19 August 2008; Date of signature: 04 March 2009

Test material

Constituent 1
Details on test material:
Sponsor's identification : H-MI ammonium salt
Description : dark red solid
Batch number : MB-1
Date received : 04 December 2008
Storage conditions : room temperature in the dark

Test animals

Species:
other: Reconstituted human epidermis

Test system

Type of coverage:
other: Topical
Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
15 minutes exposure and 42 hours post exposure incubation.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
96.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Direct MTT Reduction

The test material was not able to directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1 (attached background material). The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 96.6% after a 15-minute exposure.

The qualitative evaluation of tissue viability is given in Table 2 (attached background material).

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 OD540for the negative contol 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-diphenyltetrazolium 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 - 1α 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. 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-Ioading. 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 μl 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 96.6% 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.