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EC number: 224-169-7 | CAS number: 4223-03-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 012
- Report date:
- 2012
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)
- Remarks:
- Date of inspection: 19 to 21-07-2011 Date of Signature: 31-08-2011
Test material
- 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
Constituent 1
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 EPISKINTM 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) |
Negative Control |
1.177 |
3.551 |
4.786 |
||
4.689 |
||
Positive Control |
290.665 |
392.138 |
416.946 |
||
468.804 |
||
Test Item |
6.487 |
8.904 |
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 EPISKINTM 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.
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