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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1988-05-04 to 1988-06-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Read-across from guideline study with RL1 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Guanidine Nitrate
- Substance type: pure active substance
- Physical state: colourless crystals
- Analytical purity: 99.3 %
- Lot/batch No.: 23/03/88 GN

Method

Species / strainopen allclose all
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
Controlsopen allclose all
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:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: dissolved in DMSO, 50 μg/plate, for TA98, TA1538 both without S9-mix
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: dissolved in distilled water, 2 μg/plate, for TA100, TA1535 both without S9-mx
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: dissolved in DMSO, 50 μg/plate, for TA1537 without S9-mix
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene dissolved in DMSO, 5 μg/plate, for TA98, TA100, TA1535 all with S9-mix
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

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Positive controls validity:
valid
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
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
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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.