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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:
The study was performed between 26 July 2011 and 01 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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 the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: EU Guideline Testing of Chemicals B46
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD 439
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
3-(methacryloylamino)propyl trimethylammonium sec-C10-13-alkylbenzene sulfonate
IUPAC Name:
3-(methacryloylamino)propyl trimethylammonium sec-C10-13-alkylbenzene sulfonate
Constituent 2
Chemical structure
Reference substance name:
3-(methacryloylamino)-N,N,N-trimethylpropan-1-aminium 4-(C10-13-sec-alkyl)benzenesulfonate
EC Number:
688-159-8
Cas Number:
1024699-81-7
Molecular formula:
UVCB substance - not available
IUPAC Name:
3-(methacryloylamino)-N,N,N-trimethylpropan-1-aminium 4-(C10-13-sec-alkyl)benzenesulfonate
Constituent 3
Reference substance name:
MAPTA-ABS salt
IUPAC Name:
MAPTA-ABS salt
Details on test material:
Sponsor's identification : 1-Propanaminium, N,N,N-trimethyl-3-[(2-methyl-1-oxo-2-propen-1-yl)amino]-, 4-C10-13-sec-alkylbenzenesulfonates
Description : amber coloured slightly viscous liquid
Batch number : P1118
Purity : 78.6 w/w%
Date received : 01 July 2011
Expiry date : 01 June 2012
Storage conditions : room temperature in the dark under nitrogen

Test animals

Species:
other: reconstituted human epidermis model
Strain:
other: reconstituted human epidermis model
Details on test animals or test system and environmental conditions:
Not applicable

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
no
Amount / concentration applied:
TEST MATERIAL

The test material was applied neat.

Amount(s) applied (volume or weight with unit):
Approximately 10 µl of the test material was applied to the epidermis surface.

Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
15 minutes & 42 hour post exposure incubation
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
Area of exposure:
Approximately 10 µl of the test material was applied to the epidermis surface.

% coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.

Time after start of exposure:
15 Minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability):
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant (Xi) R38

Mean tissue viability is >50% : Non-Irritant

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Viability of cells
Remarks on result:
other:
Remarks:
Basis: mean Viability of cells. Time point: Day 6. Reversibility: other: Not applicable. Remarks: See relative mean viability below.. (migrated information)

In vivo

Irritant / corrosive response data:
The relative mean viability of the test material treated tissues was 39.6% after a 15 minute exposure.
Other effects:
No

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue which indicated that the test material did not 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(see below). The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1(see below).

The relative mean viability of the test material treated tissues was 39.6% after a 15-Minute exposure period.

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 value of the percentage viability was ≤18%. 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 percentage viability was ≤18%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was ≤18%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Item

OD540of tissues

Mean OD540of triplicate tissues

±SDof OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.654

0.660

0.040

99.1

100*

6.1

0.623

94.4

0.703

106.5

Positive Control Item

0.060

0.049

0.011

9.1

7.4

1.7

0.038

5.8

0.049

7.4

Test Item

0.304

0.261

0.044

46.1

39.6

6.6

0.263

39.8

0.217

32.9

SD=    Standard deviation

*=     The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The test item was considered to be Irritant (I) EU Risk Phrase R38.
Executive summary:
Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTM 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-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.

Methods:

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-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 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 percentage 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 39.6% after a 15-minute exposure.

Quality criteria: 

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

Conclusion:  The test item was considered to be Irritant (I) EU Risk Phrase R38.

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