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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation in bacteria:


In the key reverse gene mutation assay in bacteria performed according to OECD guideline 471, 1983, strains TA 98, TA 100TA 1535, TA l537 and TA l538 of S. typhimurium were exposed to Guanidine Nitrate, (99.3 % a.i. dissolved in sterile water), at concentrations of 8, 40, 200, 1000 and 5000 µg/plate in the first experiment and1000, 2000, 3000, 4000, 5000 µg/plate in the second experiment in the presence and absence of mammalian metabolic activation (rat liver S9-mix) using the plate co-incubation method. Guanidine Nitrate was tested up the limit concentration of 5000 µg/plate. No cytotoxicity and no increase in the number of revertants were observed in all tester strains, with or without metabolic activation.The positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed. There was no evidence of induced mutant colonies over background. This study is regarded as acceptable. This study satisfies the requirement for Test Guideline OECD 471 (1983) for in vitro mutagenicity (bacterial reverse gene mutation) data. In a supporting study, the results of the key study are fully confirmed.


Additionally supporting data from an Ames assay including the tester strain E.coli wp2uvra are available for the read-across substance guanidine monohydrochloride. Negative results were obtained with guanidine monohydrochloride in the tester strains TA 1535, TA1537, TA1537, TA98, TA100 and E. coli wp2uvra at concentrations ranging from 20 to 5000 µg/plate with and without mammalian metabolic activation.


 


Cytogenetics (Chromosome Aberrations) in mammalian cells:


In a mammalian cell cytogenetics assay similar to OECD Guideline 473, 1997 cultures of a clonal sub-line of a Chinese hamster fibroblast cell line (CHL) were exposed to a large number of chemicals in a comparative test. Treatment with Guanidine Nitrate was performed at a maximum concentration of 0.5 mg/ml (41 x 10-4M) without metabolic activation. Microscopic analysis: In the experiment no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item were close to the range of the solvent control values (below 3 % inclusive gaps). Experimentation in the presence of metabolic activation was not performed. In conclusion it can be stated that under the experimental conditions reported,i.e. without metabolic activation, Guanidine Nitrate did not induce structural chromosome aberrations as determined by the chromosome aberration test in CHL cells (Chinese hamster cell line) in vitro. The test item is considered to be non-clastogenic in this chromosome aberration test without metabolic activation when tested up to cytotoxic test item concentrations.


 


Gene mutation in mammalian cells:


In a mammalian gene mutation assay according to OECD Guideline 476, L5178 Y (mouse lympoma thymidine kinase locus) cells cultured in vitro were exposed to guanidine nitrate (> 99.8 %) in the presence and absence of mammalian metabolic activation at the following concentrations: Experiment I: 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL - with and without S9 mix Experiment II: 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL - with S9 mix Experiment II: 75.0, 150.0, 300.0, 600.0, and 900.0 µg/mL - without S9 mix Relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed at 600 µg/mL and above in the second experiment without metabolic activation following 24 hours of treatment. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.


 


Conclusion:


In conclusion the results from gene mutation assays in bacteria and mammalian cells, as well as chromosome aberration in mammalian cells are consistently negative. Thus, there is no evidence for a genotoxic potential of Guanidine Nitrate.


 


Supporting data:


Additional supporting data from the read-across substance guanidine chloride were included to demonstrate the similar toxicological profile of both substances.


 


Justification for read-across:


Guanidine hydrochloride and guanidine nitrate dissociate in aqueous media to yield the guanidine ion and the respective anion. Therefore it is reasonable to discuss the effects of the ions separately. The chloride ion is a naturally occurring essential ion in human beings with well-known metabolism and mechanisms of action as described in standard textbooks on pharmacology and physiology. As well it is found as salt in the Earth´s crust and is dissolved in seawater. Effects of guanidine hydrochloride are expected to be based primarily on the guanidine ion. The physiological processing of the guanidine ion is expected to be independent of the individual source. Therefore read-across from guanidine hydrochloride for effects of guanidine dissociated from guanidine nitrate is considered valid. This strategy is supported by a quite similar toxicological profile of both substances, as shown in acute toxicity, irritation, sensitization and genotoxic studies. For further information, please refer to the detailed justification paper attached or chapter 1.1.2 Justification for Read-across (Analogue approach) of CSR.



Short description of key information:
In vitro data for Guanidine Nitrate are available from two gene mutation studies in bacteria (Ames Tests), from a publication on in vitro chromosome aberrations and a mammalian gene mutation assay. Additionally data from a bacterial reverse mutation assay with the read-across substance guanidine monohydrochloride are available. The results are consistently negative. There is no evidence for a genotoxic potential of Guanidine Nitrate.

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988-05-04 to 1988-06-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983-05-26
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Histidine auxotroph
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: Histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (fraction of liver of male Wistar rats incuded with Aroclor 1254); prepared by the testing facility.
Test concentrations with justification for top dose:
Range finder and first experiment: with and without metabolic activation 8, 40, 200, 1000, 5000 μg/plate
Second experiment: with and without metabolic activation: 1000, 2000, 3000, 4000, 5000 μg/plate
Vehicle / solvent:
- Vehicle used: sterile distilled water
Untreated negative controls:
yes
Remarks:
equal to vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
vehicle is sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not done
- Exposure duration: at least 2 days

NUMBER OF PLATES EVALUATED: three per dose

NUMBER OF REPLICATIONS: two indipendent experiments were performed

DETERMINATION OF CYTOTOXICITY: by inspection of the background lawn
Evaluation criteria:
ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
(i) the mean negative control counts fell within the normal range of the test facility
(ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
(iii) no more than 5% of the plates were lost through contamination or some other unforseen event
EVALUATION CRITERIA
A test compound was considered to be mutagenic if:
(i) the assay was valid (see ACCEPTANCE CRITERIA)
(ii) two or three-fold increases (dependent on strain) in revertant numbers, were accompanied by significant F-statistics and dose response correlations
(iii) the positive responses described in (ii) were reproducible
Statistics:
not performed due to absence of two or three-fold infcreases (dependent on strain) in revertant numbers
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Strain TA100 was tested with Guanidine Nitrate at final concentrations of 8, 40, 200, 1000 and 5000 μg/plate in the absence and presence of S9. No signs of toxicity were observed, so the data obtained were considered acceptable as TA100 mutagenicity data for experiment 1. The same dose
range was used to treat the other strains in experiment 1, and the only sign of toxicity seen was a reduction in TA9B revertants at the top dose
in the absence of S9.
For experiment 2 the same top dose was used, and a very slight reduction in TA100 revertants at 5000 μg/plate in the absence of S9 may have
indicated some toxicity. 'The range was narrowed (to 1000-5000 μg/plate) to examine more closely those doses most likely to elicit a mutagenic
response.

COMPARISON WITH HISTORICAL CONTROL DATA:
From the data it can be seen that mean solvent control counts fell within the normal historical range, that the positive control chemicals all induced large increases in revertant numbers in the appropriate strains, and that < 5% of plates were lost, leaving adequate numbers of plates at all treatments. The study was accepted as valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
An initial toxicity range-finder experiment was carried out in TA100 only, using final concentrations of guanidine nitrate at 8, 40, 200, 1000 and 5000 μg/plate plus a solvent and positive control. No evidence of toxicity was observed in the range-finder experiment.
Remarks on result:
other: all strains/cell types tested

All Guanidine Nitrate treatments, in both experiments, yielded revertant numbers that were similar to those seen in controls. The two-fold (TA98, TA100) and three~fold (TA1535, TA1537, TA1538) increasesin revertant numbers that would be required for a clear positive response were not achieved. The results, therefore, provide no evidence that Guanidine Nitrate can induce reverse mutation in these strains of bacteria, either in the absence or presence of rat liver S9.

Conclusions:
Guanidine Nitrate is considered to be negative in the reverse gene mutation assay in bacteria (S. typhimurium strains TA 98, TA 100 TA 1535, TA l537 and TA l538), when tested up to the limit concentration of 5000 µg/plate in the presence and absence of mammalian metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, 1983, strains TA 98, TA 100TA 1535, TA l537 and TA l538 of S. typhimurium were exposed toGuanidine Nitrate, (99.3 % a.i. dissolved in sterile water), at concentrations of 8, 40, 200, 1000 and 5000 µg/plate in the first experiment and1000, 2000, 3000, 4000, 5000 µg/plate in the second experiment in the presence and absence of mammalian metabolic activation (rat liver S9-mix) using the plate co-incubation method.

 

Guanidine Nitratewas tested up the limit concentration of 5000 µg/plate. No cytotoxicity and no increase in the number of revertants were observed in all tester strains, with or without metabolic activation.The positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed.  

There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 (1983) for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no metabolic activation
GLP compliance:
no
Remarks:
study performed before implementation of GLP
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: clonal sub-line of a Chinese hamster fibroblast cell line (CHL)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's MEM (GIBCO F-11) supplemented with 10 % calf serum
- Doubling time: estimated as 18.2 h at exponential cell growth at 37 °C in a 5 % CO2 atmosphere.
Metabolic activation:
without
Test concentrations with justification for top dose:
Three different doses, including the 50 % inhibition dose
Vehicle / solvent:
DMSO 0.5 %
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
ethylnitrosurea
Details on test system and experimental conditions:
Three different doses, including the 50 % inhibition dose of each agent, which was estimated by the growth inhibition test described below, were prepared and separately added to 3-day-old cultures (about 10E5 cells/6-cm dish). Chromosome preparations were made, as a rule at 24 and 48 h, or when necessary at 6 h, after the treatment. Cells were treated with colcemid (0.2 µg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KC1 hypotonic solution for 15 min at 37 °C. The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3 times. A few drops of the suspension were then placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1 % Giemsa's buffered solution (pH 6.8) for 20 min.
The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases at the magnification of 700. Types of aberration were classified into 5 groups: chromatid gaps (g), chromatid breaks (b), chromatid or chromosomal translocation (t), ring formation (r) and fragmentation or pulverization (f). Breaks less than the width of a sister chromatid were designated as gaps in our criteria. The incidence of polyploid cells was also calculated. Untreated cells and cells treated only with solvent served as controls.
Evaluation criteria:
CHL cells commonly have less than 3.0 % cells with chromosomal aberrations. Therefore, the final judgement given to all experimental groups was as
follows. Negative (--) if less than 4.9 % of the aberration was detected even when doses of the agent were elevated to sub-lethal amounts, where almost no mitosis was observed; suspicious (+) if between 5.0 and 9.9 %, and positive if between 10.0 and 19.9 % (+), 20.0 and 49.9% (++) or more than 50.0 % (+++). When no reasonable dose response was obtained, additional experiments with different doses were carried out to confirm its reproducibility.
Key result
Species / strain:
other: clonal sub-line of a Chinese hamster fibroblast cell line (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Even at such high doses that the cells were about to be killed, no significant increases of chromosomal aberration were detected, although cytolysis as well as karyorrhexis were frequently observed.
Incidences of polyploid cells and chromosomal aberrations in control cells: No incidence was more than 2.0 %, indicating that there are no significant increases in numbers of polyploid cells or of chromosomal aberrations after treatment by solvent alone.
Remarks on result:
other: all strains/cell types tested

- name of compounds tested

- origin: Dept. of Chemistry - National Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158 (Japan)

- solvent used for dilution: DMSO (0.5 % in final)

- doses: max. effective dose: 0.50 mg/ml (41 x 10-4 M)

- % chromosome aberrations: 2 %

- time for maximal effects: 24 h

- type of aberration: gaps

- final judgement: negative

Conclusions:
Under the experimental conditions, Guanidine Nitrate did not induce chromosome aberrations when tested without metabolic activation.
Executive summary:

In a mammalian cell cytogenetics assay similar to OECD Guideline 473, 1997 cultures of a clonal sub-line of a Chinese hamster fibroblast cell line (CHL) were exposed to a large number of chemicals in a comparative test.

Treatment with Guanidine Nitrate was performed at a maximum concentration of 0.5 mg/ml (41 x 10-4 M) without metabolic activation. Microscopic analysis: In the experiment no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item were close to the range of the solvent control values (< 3 % inclusive gaps).

Experimentation in the presence of metabolic activation was not performed.

 

Positive controls induced the appropriate response: Ethylnitrosourea and 4-Nitroquinoline-N-Oxide were among the tested substances which resulted positive and showed distinct increase in cells with structural chromosome aberrations.

 

In conclusion it can be stated that under the experimental conditions reported, i.e. without metabolic activation, Guanidine Nitrate

did not induce structural chromosome aberrations as determined by the chromosome aberration test in CHL cells (Chinese hamster cell line) in vitro.The test item is considered to be non-clastogenic in this chromosome aberration test without metabolic activation when tested up to cytotoxic test item concentrations.

There was no evidence of chromosome aberration induced over background.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
This in vitro test is an assay for the detection of forward gene mutations at the autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TK+/- cells to TK-/- mutants.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Complete culture Medium: RPMI 1640 medium supplemented with 15 % horse serum (3 % HS during 4 hour treatment), 1% of 100 U/100 Pg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal.
- Selecitve Medium: Complete culture medium by addition of 5 µg/mL TFT
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I: 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL - with and without S9 mix
Experiment II: 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL - with S9 mix
Experiment II: 75.0, 150.0, 300.0, 600.0, and 900.0 µg/mL - without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I 4 hours with and without S9 mix; Experiment II 4 hours with S9 mix and 24 hours without S9 mix.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 - 15 days

SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: 4×10 E3 cells per microtiter plate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth;

OTHER EXAMINATIONS:
- Other: If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus
large colonies clastogenic effects are indicated.

Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the
corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into
consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to
differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose
dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11
(SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software.
The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups.
A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 600µg/mL and above without S9 mix following 24 hours exposure.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Guanidinium nitrate is considered to be non-mutagenic in this mouse lymphoma assay.
Executive summary:

In a mammalian gene mutation assay according to OECD Guideline 476, L5178 Y (mouse lympoma thymidine kinase locus) cells cultured in vitro were exposed to guanidine nitrate (> 99.8 %) in the presence and absence of mammalian metabolic activation at the following concentrations:

Experiment I: 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL - with and without S9 mix

Experiment II: 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL - with S9 mix

Experiment II: 75.0, 150.0, 300.0, 600.0, and 900.0 µg/mL - without S9 mix

Relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed at 600 µg/mL and above in the second experiment without metabolic activation following 24 hours of treatment. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion it can be stated that the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, Guanidinium nitrate is considered to be non-mutagenic in this mouse lymphoma assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1988-05-04 to 1988-06-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Supporting information for read-across, structural analogue approach.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 26 May 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: fra; biotin deficient; TA98 and TA 100: pKM101
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other:
Remarks:
fra; biotin deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment 1: 0, 8, 40, 200, 1000 and 5000 µg/plate
Experiment 2: 1000, 2000, 3000, 4000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: test substance is water soluble
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: at least 2 days

NUMBER OF REPLICATIONS: triplicate plates were used for testsubstance concentrations,quintuplicate plates for negative controls ,
two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: background lawn



Evaluation criteria:
Validity criteria:
(i) the mean negative control counts fell within the normal range.
(ii) the positive control chemicals induced clear increases in revertant numbers.
(iii) no more than 5% of the plates were lost through contamination or some other unforseen event.

Evaluation criteria:
A two-fold increase in TA98 or TA100 revertants, and a three-fold increase in TA1535, TA1537 or TA1538 revertants, from concurrent controls, indicates a positive response.
Statistics:
none
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: soluble
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
0, 8, 40, 200, 1000 and 5000 µg/plate - no toxicity was observed

COMPARISON WITH HISTORICAL CONTROL DATA:
all data within historical control data

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of significance.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Guanidine hydrochloride is considered to be negative in the reverse gene mutation assay in bacteria (S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538), when tested up to the limit concentration of 5000 µg/plate in the presence and absence of mammalian metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, 1983, strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Guanidine hydrochloride (98.5% a.i.) in water at concentrations of at concentrations of 8, 40, 200, 1000 and 5000 µg/plate in the first experiment and 1000, 2000, 3000, 4000, 5000 µg/plate in the second experiment in the presence and absence of mammalian metabolic activation (rat liver S9-mix) using the plate co-incubation method.

Guanidine hydrochloride was tested up to limit concentrations of 5000 µg/plate. No cytotoxicity was observed and no increase in the number of revertants were observed in all tester strains, with or without metabolic activation. The positive controls induced the appropriate responses in the corresponding strains and activity of metabolising system was confirmed.

There was no evidence of induced mutant colonies over background.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 (1983) for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Justification for type of information:
Supporting information for read-across, structural analogue approach.
Qualifier:
equivalent or similar to guideline
Guideline:
other: No guideline reported.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9; phenobarbital and betanaphthoflavone induced
Test concentrations with justification for top dose:
20 - 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: NA
Details on test system and experimental conditions:
METHOD OF APPLICATION:preincubation

Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Conclusions:
Negative in the tester strains TA 1535, TA1537, TA1537, TA98, TA100 and E. coli wp2uvra with and without mammalian metablic activation.
Executive summary:

Mutagenicity test data for guanidine monohydrochloride were published in the chemical carcinogenesis research information system (CCRIS, 2003). Negative results were obtained in the tester strains TA 1535, TA1537, TA1537, TA98, TA100 and E. coli wp2uvra in a concentration range from 20 to 5000 µg/plate, with and without mammalian metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Histidine auxotroph
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: Histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
S-9 microsomal fraction purchased from Litton Bionetics (Louisiana, MD)
Test concentrations with justification for top dose:
TOXICITY TESTS: without metabolic activation 0.0016-0.008-0.04-0.2-1-5 mg/plate
MUTAGENICITY ASSAY: without metabolic activation 0.0016-0.008-0.04-0.2-1-5 mg/plate
Vehicle / solvent:
- Vehicle used: autoclaved water from a polymetrics water filter system
Untreated negative controls:
yes
Remarks:
equal to vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
vehicle is water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Positive controls:
yes
Positive control substance:
other: aminoanthracene
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-N-nitrosoguanidine
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: one

NUMBER OF PLATES EVALUATED: three per dose

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A compound is consideed mutagenic if the following criteria are met:
- The test compound causes revertant colony counts greater than or equal to twice the spontaneous revertant rate.
- The test compound produces a correlated dose response relationship.
Statistics:
not performed
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Guanidine Nitrate is considered to be negative in the reverse gene mutation assay in bacteria (S. typhimurium strains TA 98, TA 100 TA 1535, TA l537 and TA l538), when tested up to the limit concentration of 5000 µg/plate in the presence and absence of mammalian metabolic activation.
Executive summary:

Guanidine Nitrate is considered to be negative in the reverse gene mutation assay in bacteria (S. typhimurium strains TA 98, TA 100 TA 1535, TA l537 and TA l538), when tested up to the limit concentration of 5000 µg/plate in the presence and absence of mammalian metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

There is no evidence for a genotoxic property from gene mutation assays in bacteria and mammalian cells, as well as chromosome aberration in mammalian cells. According to CLP, EU GHS (Regulation (EC) No 1272/2008), as well as Directive 67/5489 EEC Guanidine Nitrate does not need to be classified for genetic toxicity.