N-(1,1,3,3-tetramethylbutyl)acrylamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 17 January 2012 and 06 February 2012

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:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU Guideline Testing of Chemicals B46

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD 439

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19 to 21-07-2011 Date of Signature: 31-08-2011

Test material

Test material information

Constituent 1

Reference substance name:

N-(1,1,3,3-tetramethylbutyl)acrylamide (t-OAA), CAS: 4223-03-4.

IUPAC Name:

N-(1,1,3,3-tetramethylbutyl)acrylamide (t-OAA), CAS: 4223-03-4.

Details on test material:

Sponsor's identification : N-(1,1,3,3-tetramethylbutyl)acrylamide (t-OAA), CAS: 4223-03-4.
Description : White solid
Batch number : 1242736
Purity : not supplied
Date received : 08 December 2011
Expiry date : 15 April 2012
Storage conditions : room temperature in the dark

Test animals

Species:

other: reconstructed human epidermis model

Strain:

other: reconstructed 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: Not applicable

Controls:

no

Amount / concentration applied:

TEST ITEM

The test item was applied neat.

Amount(s) applied (volume or weight with unit):
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis.

Concentration (if solution):
The test item 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 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis.

% coverage:
The test item was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis.

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 item.

Time after start of exposure:
15 Minutes post exposure

SCORING SYSTEM
Quantitative MTT Assessment (percentage tissue viability):
For the test item 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 item / 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

Mean tissue viability is >50% : Non-Irritant

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: MTT reduction

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Test Item, Positive Control Item and Negative Control Item

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

The relative mean viability of the test item treated tissues was 109.4% after a 15 -Minute exposure period.

The sponsor requested the concentration ofinflammatory mediator IL-1α in the retained culture medium to be measured for confirmatory purposes.

The concentration of inflammatory mediator IL-1α in the culture medium retainedfrom the test item treated tissues was 8.904 pg/ml. This result confirmed the absence of cytotoxicity as determined in the MTT assay.

Quality Criteria

The relative mean tissue viability for the positive control treated tissueswas 7.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was 0.892 and the standard deviation value of the percentage viability was 2.2%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 4.0%. The test item acceptance criterion was therefore satisfied.

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

Item	OD540 of tissues	Mean OD540 of triplicate tissues	±SDof OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.910	0.892	0.020	102.0	100*	2.2
	0.895			100.3
	0.871			97.6
Positive Control Item	0.057	0.063	0.010	6.4	7.1	1.1
	0.074			8.3
	0.058			6.5
Test Item	0.942	0.976	0.036	105.6	109.4	4.0
	0.973			109.1
	1.013			113.6

SD =   Standard deviation

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

Determination of IL-1αConcentration in Culture Medium

In response to physical or chemical stress, keratinocytes produce and release a number of cytokines and other signalling factors which rapidly generate cutaneous inflammation. This observation allows the measurement of such keratinocyte responses to evaluate toxicological properties of chemicals in order to identify irritants and/or sensitisers. The main endpoint measured for the EPISKIN^TM Reconstructed Epidermis model is cell viability as measured by MTT reduction. However, a complimentary endpoint, IL-1α release, is incorporated for those chemicals which do not reduce tissue viability below the threshold for predicting irritancy (50%). This complimentary endpoint was used to either confirm a non-irritant result, or used to override the non-irritant result thereby improving the sensitivity of the assay.

Procedure: For test items showing a relative tissue viability of >50%, the amount of IL-1α (pg/ml) released into the tissue culture medium at the end of the 42 -hour post-exposure incubation period was measured in duplicate using R&D systems IL-1α ELISA kit DLA50 (R&D systems, Abingdon, UK). A detailed test procedure was supplied with the kit.

Interpretation of Results: The test item was considered to be an irritant if the relative tissue viability after 15 minutes exposure and 42 hours post-exposure incubation was >50% and the amount of IL-1α release was >60pg/ml.

The test item was considered to be non-irritant if the relative tissue viability after 15 minutes exposure and 42 hours post-exposure incubation was >50% and the amount of IL-1α release was ≤60pg/ml.

Appendix 1 (continued)   Determination of IL-1α Concentration in Culture Medium

Results

Item	Concentration (pg/ml)	Mean Concentration (pg/ml) (SD)
Negative Control	1.177	3.551 (±2.056)
	4.786
	4.689
Positive Control	290.665	392.138 (±91.624)
	416.946
	468.804
Test Item	6.487	8.904 (±9.393)
	0.955
	19.269

SD=   Standard deviation

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

The test item was considered to be Non-Irritant. This result was confirmed by assessment of the inflammatory mediator IL-1α.

Executive summary:

Introduction: The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed 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 reconstructed human epidermal cultures following topical exposure to the test item 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 item 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 items 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. This method was designed to be compatible with the following:

OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010)

Method B.46 of Commission Regulation (EC) No. 440/2008/EC

Method: Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 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 item treated tissues relative to negative control tissues).

Results: The relative mean viability of the test item treated tissues was 109.4% after the 15-Minute exposure period.

The sponsor requested the concentration ofinflammatory mediator IL-1α in the retained culture medium to be measured for confirmatory purposes.

The concentration ofinflammatory mediator IL-1α in the culture medium retainedfrom the test item treated tissues was 8.904 pg/ml. This result confirmed the absence of cytotoxicity as determined in the MTT assay.

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

Conclusion: The test item was considered to be Non-Irritant. This result was confirmed by assessment of the inflammatory mediator IL-1α.