Amantadine hydrochloride

Endpoint summary

Additional information

There are several, not fully reliable in vitro studies available to assess the genetic toxicity. Nevertheless, sufficient information is available when taking in account all in vitro studies collectively and when considering the reliable in vivo micronucleus study.

In vitro

Gene mutation in bacteria

In a bacterial reverse gene mutation assay, S. typhimurium was exposed to the read across test item (CAS 768 -94 -5, purity unknown) in the presence and absence of mammalian metabolic activation (Brambilla and Martelli 2009). There were no data available about the solvent and the concentrations of the test item as well as about positive controls used.

There was no evidence of mutagenicity in the Ames test using Salmonella typhimurium. The publication is only a secondary source and does not satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data (limited documentation, no data about the used bacterial strains, no data about investigated concentrations, no data about used positive controls. It is therefore classified as not assignable (reliability of 4).

The read across substance was negative in Salmonella typhimurium assay with and without metabolic activation.

Gene mutation in mammalian cells

In a mammalian cell gene mutation assay (HPRT locus), Chinese Hamster Ovary (CHO) cells cultured in vitro were exposed to the read across substance (CAS 768 -94 -5, unknown purity), in the presence and absence of metabolic activation (Brambilla and Martelli 2009). There were no data available about the solvent and the concentrations of the test item as well as about positive controls used.

There was no evidence of mutagenicity in the mammalian cell gene mutation assay. The publication is only a secondary source and does not satisfy the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data (limited documentation, no data about investigated concentrations, no data about metabolic activation system given and no data about used positive controls). It is therefore classified as not assignable (reliability of 4).

The read across substance was negative in the mammalian cell gene mutation assay with and without metabolic activation.

Cytogenicity in mammalian cells

In an in vitro mammalian chromosome aberration test, human peripheral blood lymphocytes were exposed to the read across substance (CAS 768 -94 -5, unknown purity), in the presence and absence of metabolic activation (Brambilla and Martelli 2009). The were no data available about the solvent and the concentrations of the test item as well as about positive controls used.

There was no evidence of mutagenicity in the mammalian chromosome aberration test. The publication is only a secondary source and does not satisfy the requirement for Test Guideline OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) data (limited documentation, no data about investigated concentrations, no data about metabolic activation system given and no data about used positive controls). It is therefore classified as not assignable (reliability of 4).

The read across substance was negative in the mammalian chromosome aberration test with and without metabolic activation.

Genome mutation in mammalian cells

In an in vitro mammalian cell transformation assay RLV-Fischer rat embryo cells (RLV/RE) were exposed to the test item (unknown purity), at concentrations of 1 and 2 µg/mL for 9 days (Price et al. 1979) in the absence of metabolic activation. The positive controls induced the appropriate responses.

Without metabolic activation the test item was negative under the experimental conditions of this assay.

Cytotoxicity was observed at a concentration of 1 µg/mL

The study is classified as acceptable (reliability 2).

There was no evidence for induction of genome mutation, as determined in the cell transformation assay in the absence of metabolic activation.

In vivo

Chromosome aberration

In a CF-1 mouse bone marrow micronucleus assay, performed according to GLP and similar to OECD 474, seven males per dose were treated intraperitoneal on three consecutive days with the test item (98% a.i.) at doses of 15, 30 or 60 mg/kg bw (Kaefer et al., 2010). Bone marrow cells were harvested on day 4. The vehicle was physiol. saline. As positive control cyclophosphamide was used.

Treated animals expressed no toxic reactions. None of the treated animals died.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any dose level. The positive control induced the appropriate response.

The study is classified as acceptable (reliability 2), but does not fully satisfy the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data (testing in single sex, limited documentation).

Under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Summary: In the Ames test, the read across substance was shown not mutagenic in Salmonella with or without metabolic activation. The read across substance was also negative in the mammalian cell gene mutation assay with or without metabolic activation. In the mammalian chromosome aberration test using human peripheral blood lymphocytes the read across substance did not induce chromosomal aberrations in the presence and absence of metabolic activation. In a mouse bone marrow micronucleus assay no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow was observed after treatment with the test item itself.

Conclusion: The read across substance showed negative effects in bacterial test systems and different in-vitro test systems in mammalian cells. No mutagenic effects of the test item were observed in an in vivo mammalian test system. Additionally, a transformation assay gave no indication on genome mutational potential of the test item itself.

Short description of key information:
in vitro
Gene mutation in bacteria
Read across substance: CAS 768-94-5 (Amantadine): Ames test with S. typhimurium with and without metabolic activation: negative (Brambilla and Martelli 2009)

Gene mutation in mammalian cells:
Mammalian cell gene mutation assay:
Read across substance: CAS 768-94-5 (Amantadine): Chinese hamster Ovary (CHO) cells with and without metabolic activation: negative (Brambilla and Martelli 2009)

Cytogenicity in mammalian cells:
Mammalian chromosome aberration test:
Read across substance: CAS 768-94-5 (Amantadine): Human peripheral blood lymphocytes with and without metabolic activation: negative (Brambilla and Martelli 2009)

Genome mutation in mammalian cells:
Mammalian cell transformation assay:
RLV-Fischer rat embryo cell system (RLV/RE) without metabolic activation: negative (Price et al., 1979)

in vivo
Cytogenicity in mammalian animals:
Mammalian chromosome aberration test: Mammalian Erythrocyte Micronucleus Test
Mouse CF-1, males, i.p. injections: negative (GLP, OECD 474, Kaefer et al., 2010)

Conclusion: The substance was not mutagenic in bacteria and in mammalian cell culture. The substance was not mutagenic in a test with mammals.

Endpoint Conclusion:

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data for genetic mutagenicty are reliable and suitable for the purpose of classification under Directive 67/548/EEC. As a result the substance is not warranted to be classified for mutagenicity under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data for genetic mutagenicity are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. As a result the substance is not warranted to be classified for mutagenicity, under Regulation (EC) No.1272/2008, as amended for the 2nd time in Commission Regulation (EU) No. 286/2011.