N-methylisopropylamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

other: In vitro Skin Corrosion: Human Skin Model Test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 431, April 13, 2004 (“In vitro Skin Corrosion: Human Skin Model Test”)

Deviations:

no

Principles of method if other than guideline:

In addition to the guideline mentioned above, the study conduct followed the test strategy for determination of a corrosive property as given in the OECD Guideline for Testing of Chemicals No. 404, April 24, 2002 (“Acute Dermal Irritation/Corrosion”).

Test principle of the EpiDermTM Corrosivity-Test:
The EpiDermTM Corrosivity-Test is an in vitro test procedure used for the detection of the corrosive potential of a test substance.
Corrosive materials are identified by their ability to produce a decrease in cell viability, as determined, for example, by using the MTT reduction assay, below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.
The test material was applied unchanged topically to a three-dimensional human skin model comprising at least a reconstructed epidermis with a functional stratum corneum for 3 minutes and 1 hour. Negative and positive controls were added.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-methylisopropylamine

EC Number:

225-266-7

EC Name:

N-methylisopropylamine

Cas Number:

4747-21-1

Molecular formula:

C4H11N

IUPAC Name:

methyl(propan-2-yl)amine

Details on test material:

- Name of test material (as cited in study report): N-Methylisopropylamine
- Lot/batch No.: 8078/06/056
- Physical state: liquid
- Analytical purity: > 99.9 area-% (analytical report No.: 07L00393)
- Stability: The stability under storage conditions over the study period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.

Test animals

Species:

other: not applicable

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

The test was conducted using the EpiDerm™ 200 Kit which consisted of 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm², cultured in Millicells® with a diameter of 1 cm.
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

undiluted

Duration of treatment / exposure:

Three minutes and one hour

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

PRETEST [Direct MTT Reduction]
The decrease in cell viability as indicator for corrosivity was determined by using the MTT reduction assay (i.e. [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide].
Approximately 50 μL test item was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at 37 °C for 55 - 65 minutes. A negative control (50 μL of doubly distilled water) was tested concurrently. If the MTT solution colour or in case of water-insoluble test substances the border to the water-phase turns blue/purple, the test item is presumed to directly reduce MTT. The direct reduction of MTT by a test item interferes with the colour density produced by metabolic capacity of the tissue and would falsify the test results. In case that direct MTT reduction occurs one freeze-killed control tissue per exposure time is treated with, each, the test article and the negative control, in the same way as described below, additionally.

MAIN TEST [EpiDermTM Assay]
On day of receipt EpiDermTM tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-item application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used. Where applicable, one killed tissue per exposure time is treated to control for direct MTT reduction. Fifty microliter (50 μL) of undiluted test item were applied using a pipette. Control tissues were concurrently treated with 50 μL of doubly distilled water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC) or test substance (killed tissue control, KC). The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of treatment and were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were again incubated for 3 hours. After incubation, the tissues were washed with PBS and the precipitated blue Formazan product produced by the tissues was extracted with Isopropanol over night at room temperature. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 3 microtiter wells filled with Isopropanol for each microtiter plate.

EVALUATION
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) together with the respective exposure time is used for evaluating whether or not a test material is corrosive.The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test is considered.
Assay acceptance criterion for the negative control (NC):
The absolute OD570 of the negative control tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay.
Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0.
Assay acceptance criterion for the positive control (PC):
Potassium hydroxide as 8.0 normal ready made solution is used as positive reference. A 3-minute treatment with 8.0 n KOH usually reveals a mean relative tissue viability of ~20%. An assay is acceptable if mean relative tissue viability of the 3 min positive control is ≤ 30%.
Assay acceptance criterion for tissue variability:
For every treatment, 2 tissues are treated in parallel. The inter tissue variability is considered to be acceptable if the difference of the OD570 values of the two tissues is ≤ 0.3.
Assay acceptance criterion for killed controls (KC):
The OD570 of the killed control tissues treated as negative control should be ≤ 0.35.

PREDICTION OF CORROSIVITY
A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
mean tissue viability (% negative control) => Interpretation
- after 3 minutes: < 50 <=> Corrosive
- after 3 minutes: ≥ 50 and after 1 hour: < 15 <=> Corrosive
- after 3 minutes: ≥ 50 and after 1 hour: ≥ 15 <=> Non-corrosive

HISTORICAL DATA
Historical ranges for negative controls, positive controls and viability were included in the study report.

Results and discussion

In vivo

Results

Irritation parameter:

other: EpiDerm Skin Corrosivity Test

Basis:

other: comparison of optical density between treated and control tissue samples.

Time point:

other: 3 minutes and 1 hours

Reversibility:

other: not applicable

Remarks on result:

other: N-Methylisopropylamine showed a corrosive potential in the EpiDerm Skin Corrosivity Test under the test conditions chosen.

Other effects:

- After 3 minutes of exposure, a mean optic density (OD) for the test item-treated skin samples of 0.359 was determined, versus 2.225 for the negative control, indicating a cell viability of 10% when compared to control (100%).
- After 1 hour of exposure, a mean optic density (OD) for the test item- treated skin samples of 0.157 was determined, versus 2.328 for the negative control, indicating a cell viability of 7% when compared to control (100%).
- For the positive control, mean OD after 3 minutes and 1 hour was 0.621 (viability 27%) and 0.235 (viability 10%), respectively. The corresponding cell viabilities confirmed the adequate performance of the test and the validity of the results.

Any other information on results incl. tables

Test material	Optical density (OD) after exposure for 3 minutes
	OD570 [tissue 1]	OD570 [tissue 2]	OD570 [killed controls KC]	Mean OD570	Mean OD570 corrected for KC	Viability [% of NC]
Negative control [NC]	2.280	2.171	0.180	2.225	-	100
Test Item	0.221	0.497	0.319	0.359	0.220	10
Positive control [PC]	0.550	0.693	-	0.621	-	27
	Optical density (OD) after exposure for 1 hour
Negative control [NC]	2.282	2.375	0.246	2.328	-	100
Test Item	0.145	0.168	0.138	0.157	-	7
Positive control [PC]	0.215	0.218	-	0.235	-	10

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information