D-α-methylbenzylamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

other: in vitro

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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: see 'Remark'

Remarks:

GLP and guideline compliant study. Study was performed with the Racemat (DL-alpha-methylbenzylamine). Read across is done to D-alpha-methylbenzylamine, as the substance is expected to behave similar to the racemat with regard to irritation properties. For read-across justification please refer to IUCLID section 13.

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:

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

Version / remarks:

(13 April 2004)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

(24 April 2002)

Deviations:

no

Test guideline 3

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)

Test material

Test material information

Constituent 1

Reference substance name:

DL-α-methylbenzylamine

EC Number:

210-545-8

EC Name:

DL-α-methylbenzylamine

Cas Number:

618-36-0

IUPAC Name:

1-phenylethanamine

Test material form:

other: liquid

Test animals

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.

Test system

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.

Results and discussion

In vivo

Results

Results 1

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

Results 2

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.

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information