Benzenemethanaminium, N,N,N-trimethyl-, 2-hydroxy-2-methylpropanoate (1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 2012 to 02 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and gudeline study

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:

other: OECD TG No. 431, April 13, 2004 “In vitro Skin Corrosion: Human Skin Model Test”

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD TG No. 439, July 22, 2010 “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method"

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008, Part B: Methods for the determination of toxicity and other health effects: In Vitro Skin Corrosion: Human Skin Model Test; Official Journal of the European Union, No. L 142

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 761/2009 of 23 July 2009: Part B: Methods for the determination of toxicity and other health effects: In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test; Official Journal of the European Union, No. L 220

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

benzyltrimethylazanium 2-hydroxy-2-methylpropanoate

EC Number:

805-580-6

Cas Number:

1431696-36-4

Molecular formula:

C14 H23 O3 N

IUPAC Name:

benzyltrimethylazanium 2-hydroxy-2-methylpropanoate

Test material form:

other: solid

Details on test material:

- Purity: The test substance has been characterized analytically.
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- pH-value: Ca. 7 (undiluted test substance, moistened with water)

Test animals

Species:

other: reconstructed human epidermal model EpiDermTM

Strain:

not specified

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 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. 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:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

66 mg per skin model

Duration of treatment / exposure:

Corrosion test: 3 min and 1 h
Irritation test: 1h and post-incubation period of 42h

Observation period:

Not applicable.

Number of animals:

Number of skin models:
Corrosion test: 2
Irritation test: 3

Details on study design:

Corrosion test:
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance 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. 25 μL de-ionized water was applied first. As the test substance could not be applied with a sharp spoon, a metal pin was covered with ca. 66 mg of the solid ground test material and was applied with direct contact to the wetted tissue. 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). 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.

Irritation test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. 25 μL sterile PBS was applied first. As the test substance could not be applied with a sharp spoon, a metal pin was covered with ca. 66 mg of the solid ground test material and was applied with direct contact to the wetted tissue. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in 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.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: viability

Value:

102

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 min. Max. score: 0.0. Remarks: Score in % viability (corrosion test) - 100% viability indicates absence of irritation/corrosion.. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: viability

Value:

93

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 h. Max. score: 0.0. Remarks: Score in % viability (corrsion test). (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: viability

Value:

113

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 43h. Max. score: 0.0. Remarks: Score in % viability (irritation test). (migrated information)

In vivo

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria it was concluded, that the substance does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU