DL-α-methylbenzylamine

Endpoint summary

Description of key information

Skin: The test item shows corrosive potential in the EpiDerm™ skin corrosion test under the test conditions chosen.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-07 to 2011-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

(13 April 2004)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

(24 April 2002)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

BIS (In Vitro) Human Skin Model Test (30 May 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: NA (in vitro test)

Strain:

other: EpiDerm human epidermis model

Details on test animals or test system and environmental conditions:

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the humanepidermis. 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. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Type of coverage:

other: Human epidermis skin model

Preparation of test site:

other: Human epidermis skin model

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

3 min or 1 hour

Observation period:

NA

Number of animals:

NA (in vitro test)

Details on study design:

DIRECT MTT REDUCTION
To assess the ability of the test material to directly reduce MTT a pretest was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. In case that direct MTT reduction occurred, one freeze-killed control tissue per exposure time was treated with, each, the test article and the negative control.

BASIC PROCEDURE
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. In addition, one killed tissue per exposure time was treated with the test substance and NC, respectively, in order to detect direct MTT reduction. Fifty microliter (50 μL) of the undiluted liquid test substance was applied. Control tissues were concurrently applied with 50 μL of de-ionized 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 the application treatment. Rinsed tissues 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 incubated for 3 hours.

After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with Isopropanol for each microtiter plate.

REMOVAL OF TEST SUBSTANCE
- Washing: Yes with PBS
- Time after start of exposure: 3 min and 1 hour

SCORING SYSTEM:
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.

HISTORICAL CONTROL DATA
The historical control values of negative controls mean OD570nm: 1.78 (3min) ; 1.772 (1hr) and positive controls mean OD570 nm:0.356 (3min) ; 0.172 (1hr); Viability: 20.17% (3min), 9.81 % (1hr), gathered over an appropriate time period (March2009-2011), are used to derive suitable acceptance criteria for the test system and to demonstrate the reproducibility of results and robustness of the procedures.

Irritation parameter:

other: loss of viability

Basis:

mean

Time point:

other: 1 hour

Score:

12

Reversibility:

other: NA

Remarks on result:

other: Score in % Viability

Irritation parameter:

other: loss of viability

Basis:

mean

Time point:

other: 3 min

Score:

57

Reversibility:

other: NA

Remarks on result:

other: Score in % Viability

Irritant / corrosive response data:

Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water. 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%.

Detailed Results

Negative Control:
3 min mean OD 570nm: 1.882; Viability 100%
1 hr mean OD 570nm: 1.899; Viability 100%

Test item:
3 min mean OD 570nm: 1.065; Viability 57%
1 hr mean OD 570nm: 0.228; Viability 12%

Positive Control:
3 min mean OD 570nm: 0.420; Viability 22%
1 hr mean OD 570nm: 0.159; Viability 8%

Due to the ability of the test substance to reduce MTT directly, a KC was applied in parallel. However, the result of the KC did not indicate an increased MTT reduction (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation.

Interpretation of results:

corrosive

Remarks:

Migrated information

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of 1-Phenylethylamine (racemat) to cause dermal corrosion was assessed by a single topical application of 50 μL of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™).

Two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability. The EpiDerm™ skin corrosion test showed the following results: The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing. Viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 57%.

Viability of the test-substance treated tissues determined after an exposure period of 1 hour was 12%. Based on the observed results and applying the evaluation criteria it was concluded, that 1-Phenylethylamine shows a corrosive potential in the EpiDerm™ skin corrosion test under the test conditions chosen.

Justification for selection of skin irritation / corrosion endpoint:
GLP and guideline compliant study.

Effects on skin irritation/corrosion: corrosive

Justification for classification or non-classification

DL-alpha-methylbenzylamine is included in Annex VI of Regulation 1272/2008/EC with the following classification:

 

Table 3.1: corrosive to skin: cat. 1B

Table 3.2: R34

 

Based on the result of the in vitro skin corrosion test (Viability 12% after 1 hour ), DL-alpha-methylbenzylamine needs to be classified as corrosive to the skin and eyes according to Directive 67/548/EEC (C, R35) and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 (Category 1B, H314: Causes severe skin burns and eye damage).