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

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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.

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)

Remarks:

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

Species:

other: reconstructed human epidermis model

Strain:

other: reconstructed human epidermis model

Details on test animals or test system and environmental conditions:

Not applicable

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

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 27 March 2012 and 29 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

no guideline available

Principles of method if other than guideline:

The protocol followed was considered to be a reliable alternative to the in vivo rabbit Draize eye irritation test in a pre-validation study. This study, using human derived keratinocytes which form a corneal epithelial tissue reconstruct, has been recommended by ECVAM for inclusion in a formal international validation study designed to offer a stand alone replacement to the in vivo test. Validation is expected to commence in 2010.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19 - 21 July 2011 Date of Signature: 31-August-2011

Species:

other: Reconstructed Human Corneal Model

Strain:

other: Reconstructed Human Corneal Model

Details on test animals or tissues and environmental conditions:

Not applicable

Vehicle:

other: No vehicle used

Controls:

no

Amount / concentration applied:

TEST ITEM

-The test Item was applied neat.

-Amounts(s) applied (volume or weight with unit):
Triplicate tissues were treated with 30 mg of the test item.

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

VEHICLE
No vehicle used.

Duration of treatment / exposure:

10 Minutes.

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

TEST SITE
-Area of exposure:
Triplicate tissues were treated with 30 mg of the test item.

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

-Type of wrap used:
None used.

REMOVAL OF TEST ITEM
-Washing (if done):
At the end of the relevant exposure period, each tissue insert was rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS). Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper.

-Time after start of exposure:
10 Minutes post exposure.

SCORING SYSTEM:
The relative mean tissue viability (percentage of the negative control) was calculated as follows:

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

The mean tissue viability for the test item was compared to the respective untreated negative control and classified according to the following:

Tissue viability <60 = Irritant
Tissue viability ≥60 = Non-Irritant

Irritation parameter:

other: viability of cells

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean viability of the test item treated tissues after a 10 Minute exposure period was 92.1%

RESULTS

Assessment of Direct Test Material Reduction of MTT

The test material was not able to directly reduce MTT.

Assessment of Eye Irritation Potential

The mean OD₅₄₀ values and mean viabilities for each treatment group are given in Table 1.

The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 92.1%.

It was considered unnecessary to proceed with tissue histopathology.

Assay Acceptance Criterion

The quality criterion required for the acceptance of results in the test was satisfied.

Table 1          Assessment of Eye Irritation Potential – Viability of HCE Tissues

Item	OD540of Individual Tissue	Mean OD540	Relative Mean Viability (%)
Negative Control	0.943	0.961	100*
	0.978
Positive Control	0.206	0.182	18.9
	0.157
Test Item	0.900	0.885	92.1
	0.870

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

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

According to the study plan followed the test item was considered to be a Non-Irritant.

Executive summary:

Introduction. The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic reconstructed Human Corneal Epithelium model (HCE, SkinEthic Laboratories,,) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods. The experimental design of the study consists of a test for direct reduction of MTT (3‑[4,5‑dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test item followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test item for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 2% w/v Sodium Dodecyl Sulphate (SDS) served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD₅₄₀). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test item was classified according to the following criteria:

If the percentage relative mean tissue viability was ≥60% the test item was considered to be non‑irritant.

If the percentage relative mean tissue viability was <60% the test item was considered to be an irritant.

Results. The relative mean viability of the test item treated tissues after a 10‑Minute exposure period was 92.1%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criterion. The quality criterion required for acceptance of results in the test was satisfied.

Conclusion. According to the study plan followed the test item was considered to be a Non-Irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 was tested in the EpiSkin in-vitro skin irritation assay using reconstructed human epidermis, which has been validated as an accepted alternative to animal testing. Irritation is measure using MTT to assess the viability of the tissue. The relative mean viability of the test item treated tissues was 109.4% after the 15-Minute exposure period, which indicated the test substance was non irritating to skin. In addition the concentration of inflammatory mediator IL-1α in the culture medium retained from the test item treated tissues was 8.904 pg/ml. This result confirmed the absence of cytotoxicity as determined in the MTT assay.

 

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

Eye Irritation

N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 was tested in the Bovine Corneal Opacity (BCOP) test, the in-vitro irritancy score at 0.4 was even lower than the negative control which was 3.4. This indicated that the test substance was not an ocular corrosive or sever irritant. The BCOP test has not been validated to indicate a lack of irritancy, classification as an eye irritant may still be possible, although the extremely low irritancy score makes this unlikely. 

As N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 is only imported in polymer it was not felt to be justified to perform and eye irritation study in rabbits. Therefore a valid but not fully validated for classification purposes test was performed. N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 was tested in the Skin Ethic HCE (Human corneal epithelial) test, using reconstructed human corneal tissue. The viability of the tissue being assessed using MTT.The test substance was classified according to the following criteria:

If the percentage relative mean tissue viability was ≥60% the test item was considered to be non‑irritant.

If the percentage relative mean tissue viability was <60% the test item was considered to be an irritant.

The relative mean viability of the test item treated tissues after a 10‑Minute exposure period was 92.1%.

According to the study plan followed theN-(1,1,3,3-tetramethylbutyl)acrylamidewas consideredto be a Non-Irritant to eyes.

Justification for selection of skin irritation / corrosion endpoint:

This study is a validated none animal test method, which showed no indication of skin irritation in reconstructed human epidermis.  The study is GLP and Klimisch validity 1.

Justification for selection of eye irritation endpoint:

This study using reconstructed human corneal epithelium, showed that N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4 was not classified as an eye irritant.  This test is valid but has not yet been fully validated but was done to GLP and is Klimisch1 validity.  This is also true for the BCOP study which is validated for severe irritants, this study also showed no evidence of eye irritation.

Justification for classification or non-classification

N-(1,1,3,3-tetramethylbutyl)acrylamide CAS 4223-03-4, was found not to be a skin irritant in the Episkin test and not to be a severe eye irritant in the BCOP and not to be an eye irritant in the Skin Ethic Humean Corneal Epithelium test. Based on these in-vitro /ex-vivio tests N-(1,1,3,3-tetramethylbutyl)acrylamide is not classified as a skin or eye irritant based on the EU CLP (GHS) criteria.