Methenamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Methenamine revealed no mutagenic effects in the Ames tests if tested up to the recommended limit concentration of 5000 µg/plate. It was weakly positive in bacterial gene mutation assays at extremely high concentrations (>= 10000 µg/plate) and in an in vitro chromosomal aberration assay in V79 cells at severely cytotoxic doses indicating some genotoxic potential in vitro test systems with bacteria and mammalian cells at very harsh test conditions.

 

A modified in vitro mouse lymphoma assay showed no mutagenicity of methenamine with and without metabolic activation. Although the study cannot be assigned to a reliability class as no complete test report is available, test results are in line with results from an in vivo dominant lethal assay (DLA). In this DLA, no indication of germ cell mutations were found after treatment of mice with different methenamine concentrations. It can therefore be concluded that methenamine does not cause gene mutations in mammalian cells. For this reason, a further in vitro gene mutation study in mammalian cells is not necessary.

 

Conclusion

In conclusion, methenamine is not considered to be of concern with regard to genetic toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

public available literature (non GLP, no guideline)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon.

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S 9 mix

Test concentrations with justification for top dose:

0, 200, 1000, 5000 µg/plate

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-ophenylenediamine

Remarks:

TA98 without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA98 and TA100 with S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 without S9 mix

Details on test system and experimental conditions:

preincubation method.

Evaluation criteria:

2-fold increase of revertants compared to negative control.

Statistics:

standard deviation

Key result

Species / strain:

S. typhimurium TA 1538

Remarks:

Not tested. This strain was tested in the other WoE Bacterial Reverse Mutation Assay (see IUCLID section 7.6.1)

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Not tested. This strain was tested in the other WoE Bacterial Reverse Mutation Assay (see IUCLID section 7.6.1)

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Not tested. This strain was tested in the other WoE Bacterial Reverse Mutation Assay (see IUCLID section 7.6.1)

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

no additional information.

Conclusions:

Methenamine is not mutagenic in Ames test up to limit concentration of 5000 µg/plate.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98 and TA100 of S. typhimurium were exposed to methenamine in water at concentrations of 0, 200, 1000, 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix). Methenamine was tested up to the limit concentration of 5000 µg/plate.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background at any test concentration with methenamine. Consequently, methenamine is evaluated to be not mutagenic in the Ames test if tested up to the limit concentration of 5000 µg/plate.

This study is classified as acceptable. This study was conducted under equivalent or similar conditions compared to the requirements of EU Method B.13/14 and OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: public available literature (non GLP, no guideline)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Principles of method if other than guideline:

Sister chromatid exchange was measured additionally

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable, chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

0.1, 1, 10, 50 mmol/L

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

incubation time: 20 h (Chromosome aberration), 30 h (Sister chromatid exchanges)

Evaluation criteria:

100 metaphases from two replicates were evaluated.

Statistics:

Student t-test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strong cytotoxic effect at a concentration of 50 mmol/L

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Chromosome aberrations:
In the highest analysable concentration of 10 mmol/l 7% aberrant cells (without gaps) were induced compared to 2% in control cultures. Higher doses were strongly cytotoxic.
Sister chromatid exchanges:
Slightly increased SCE frequencies were described after treatment of V79 cells with 10 and 50 mmol/l methenamine.
Historical control data and other critical parameters at high concentrations (osmolarity, pH) not reported.

Conclusions:

Methenamine induced chromosome aberrations in the highest analysable concentration of 10 mmol/L. Slightly increased Sister Chromatid Exchange frequencies were also described after treatment of V79 cells with 10 and 50 mmol/L methenamine.

Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration (CA) and Sister Chromatid Exchanges (SCE)), V79 cell cultures were exposed to methenamine in water at concentrations of 0, 0.1, 1, 10, 50 mmol/L without metabolic activation.

Methenamine was tested up to cytotoxic concentration. In the highest analysable concentration of 10 mmol/l 7% aberrant cells (without gaps) were induced compared to 2% in control cultures. Higher doses were strongly cytotoxic. Only one experiment without S-9 mix was done. In the same publication slightly increased SCE frequencies were described after treatment of V79 cells with 10 and 50 mmol/l methenamine. Positive controls induced the appropriate response. There was a concentration related positive response of Chromosome aberration and SCE induced over background.

This study is classified as acceptable but due to missing data about cytotoxicity a final evaluation is difficult. This study satisfies the requirement for Test Guideline (OECD 473, EU B.10 and in vitro sister chromatid exchange assay in V79 cells) for in vitro cytogenetic mutagenicity data.

Genetic toxicity in vivo

Description of key information

No mutagenic effects were noted in vivo tests. Neither an in vivo chromosomal aberration test in mice nor a dominant lethal test in mice revealed any genotoxic potential in vivo. Consequently, methenamine is not considered to be of concern with regard to genetic toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro assays with bacteria

In a bacterial gene mutation assay weak positive effects with and without S-9 mix were noted for tester strains TA 97, TA 98 and TA 100 (approx. 2-fold increases compared to control values) only in high concentrations from 10 000 μg/plate upwards (Zeiger et al., 1992). The approximately 2-fold increases of the mutation rates (compared to the control value) were observed only at clearly higher concentrations than 5000 μg/plate, the maximum test concentration recommended by the guideline (Shimizu et al., 1985). The relevance of such findings is questionable at such high concentrations as indirect effects might be responsible for the biological response noted.For tester strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 up to 5000 μg/plate negative results were found with and without S-9 mix (Andrews et al., 1980; Crebelli et al., 1984; Takahashi, 1993).

 

In vitro assays with mammalian cells

Girmanova et al. (1991) reported on a positive chromosomal aberration assay with V79 cells: In the highest analysable concentration of 10 mmol/l 7% aberrant cells (without gaps) were induced compared to 2% in control cultures. Higher doses were strongly cytotoxic. Only one experiment without S-9 mix was done. In the same publication slightly increased SCE frequencies were described after treatment of V79 cells with 10 and 50 mmol/l methenamine. No historical control data are given to allow a final assessment of the effects noted.

In a poorly documented chromosomal aberration assay with HeLa cells negative results were found up to concentrations of 1 mmol/l; higher tested doses induced strong cytotoxic effects (Baldermann et al., 1967). Information on the use of S9 is not given.

Dooley et al. (1985) reported in an abstract without detailed data on a negative mouse lymphoma assay with methenamine under addition of formaldehyde dehydrogenase and NAD+ to the test system.

 

In vivo assays with mammals

Two in vivo chromosomal aberration tests with mouse bone marrow cells were negative (Vujosevic et al. 1986). An in-vivo chromosomal aberration test with mouse bone marrow cells led to a negative result after single oral doses up to 618 mg/kg (corresponding to 1/3 of LD50-value). Sampling times were 6 h, 12 h, and 24 h after single treatments. Another in vivo chromosomal aberration test with mouse bone marrow cells led also to a negative result after repeated oral doses. Doses up to 618 mg/kg bw were given five times with intervals of 24 h; sampling time was 6 h after last administration. No information about clinical symptoms or cytotoxic effects is given by the authors. However, from the toxicokinetic data available it can be concluded that the substance was available at the target organ.

A dominant lethal assay with mice led to a negative result after single i.p. doses of up to 10 g/kg methenamine. A second trial, in which oral doses of 25 g/kg (maximum tolerated dose) were administered, is not valid, because of higher frequencies of live implants in treated animals than in control animals. No positive control substances were included. Methenamine is not mutagenic in a dominant lethal assay in mice even tested at clear systemic toxic doses of up to 25 g/kg bw.

 

Conclusion: As no aneugenic or clastogenic effects were found in in vivo studies, methenamine is considered to be non-genotoxic.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation No 605/2014.