Amantadine hydrochloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method: 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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Amantadine hydrochloride

EC Number:

211-560-2

EC Name:

Amantadine hydrochloride

Cas Number:

665-66-7

Molecular formula:

C10H17N.ClH

IUPAC Name:

adamantan-1-amine hydrochloride

Details on test material:

- Name of test material (as cited in study report): Adamantylamin hydrochlorid
- Analytical purity: 99.7 g/100 g
- Test-substance No.: 11/0506-1
- Lot/batch No.: 20110315
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage stability: The stability under storage conditions over the study period was guaranteed.
- Other:
pH-value: 5.6 at 20°C (10% aqueous solution)

Test animals

Species:

other: in vitro test on three dimensional human epidermis model (EpiDermTM model which consists of normal human-derived epidermal keratinocytes cultured to form a multi layered, highly differentiated model of the human epidermis.)

Strain:

other: not applicable

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (in vitro test)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL bulk volume (about 20 mg)

Duration of treatment / exposure:

3 minutes and 1 hour

Observation period:

not applicable (in vitro test)

Number of animals:

not applicable (in vitro test)

Details on study design:

Test system:
- In vitro test system on three dimensional human epidermis model. The EpiDermTM model consists of normal, human-derived epidermal keratinocytes 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.

Material and technical equipment:
- EpiDerm™ 200 kit: MatTek Corp., Ashland MA, USA containing 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® diameter 1 cm.

Controls:
- Negative control (NC): 50 µL of de-ionized water
- Positive control (PC): 8 N potassium hydroxide solution (Sigma-Aldrich, Munich, Germany)

Experimental procedure:
- First 25 µL de-ionized water was applied. Thereafter, a bulk volume of 25 µL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.
- After application of the test material to the stratum corneum surface of the EpiDermTM tissue the induced cytotoxicity (= loss of viability) is measured by a colourimetric assay.
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow coloured water soluble MTT to the insoluble blue coloured formazan. After isopropanol extraction of the formazan from the tissues, the optical densitiy of the extract is determined spectrophotometrically. Optical density of the extracts of test substance treated tissues is compared to negative control values and expressed as relative tissue viability.

Evaluation criteria:
- The test substance is considered as corrosive to the skin, if the mean relative tissue viability (% of negative control) after 3 min treatment 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%.
- Although the method is not finally validated for categorizing the severity of corrosion according to certain classification and labeling systems, it is suggested to use the most stringent category for test substances leading to viabilities below 50% after 3 min treatment.

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. The mean OD570 of the NC should not exceed 2.5.
- Assay acceptance criterion for the positive control (PC): Potassium hydroxide as 8.0 N 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.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: viability (%)

Value:

85

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 min. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Corrosion in vitro test. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: viability (%)

Value:

13

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 h. Max. score: 100.0. Reversibility: other: not applicable. Remarks: Corrosion in vitro test. (migrated information)

In vivo

Irritant / corrosive response data:

The test substance is not able to reduce MTT directly.
- Viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 85%.
- Viability of the test-substance treated tissues determined after an exposure period of 1 hour was 13%.

Any other information on results incl. tables

Table 2: Corrosion test

Test substance		Expsosure: 3 min			Exposure: 1 hour
		Tissue 1	Tissue 2	Mean	Tissue 1	Tissue 2	Mean
NC	Mean OD570	1.918	1.922	1.920	1.930	1.774	1.852
	Viability (% of NC)	99.9	100.1	100	104.2	95.8	100
Test Substance	Mean OD570	1.708	1.569	1.639	0.244	0.244	0.244
	Viability (% of NC)	89.0	81.7	85	13.2	13.2	13
PC	Mean OD570	0.399	0.293	0.346	0.166	0.217	0.192
	Viability (% of NC)	20.8	15.2	18	9.0	11.7	10

NC: Negative control (De-ionized water)

PC: Positive control (8 N potassium hydroxide solution)

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the observed results and applying the evaluation criteria, it was concluded, that the test substance shows a corrosion potential in the EpiDerm™ skin corrosion test under the test conditions chosen.