Diguanidinium carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 1998-08-03 to 1998-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study without deviations, performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

from 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

from 29 December 1992

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diguanidinium carbonate

EC Number:

209-813-7

EC Name:

Diguanidinium carbonate

Cas Number:

593-85-1

Molecular formula:

CH5N3.1/2CH2O3 (one guanidine species, as denoted in the ESIS database) CH5N3.CH5N3.CH2O3 (as in the crystalline from, basis for the molecular weight 180.1658 g/mol as given below)

IUPAC Name:

bis(amino(imino)methanaminium) carbonate

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Guanidine Carbonate Pure
- Substance type: Salt with organic kation and inorganic anion
- Physical state: White crystalline powder
- Analytical purity: 99.7 %
- Impurities: Sulphate ash <0.05 %, heavy metals calculated as lead, <10 ppm (mg/L), iron 1 ppm, lead <1 ppm, arsenic <3 ppm
- Purity test date: 1998-06-09
- Lot/batch No.: 1867 of Agrolinz Melamin GmbH, St.-Peter-Strasse 25, A-4021 Linz
- Expiration date of the lot/batch: Not specified but considered stable under conditions of storage for at least 5 years
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

Histidine for Salmonella
Tryptophan for E. Coli

Species / strain

Species / strain / cell type:

other: TA97a, TA98, TA100, TA 102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

microsomal fraction of homogenised livers of rats treated once with 500 mg/kg of Aroclor 1254

Test concentrations with justification for top dose:

First experiment: 5000, 166, 556, 185 and 62 µg/plate
Second experiment: 5000, 1667, 556, 185, and 62 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: not reported

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: 4-Nitro-o-phenylene-diamine (TA97a, TA98), Sodium-azide (TA100, TA135), t-Butyl-hydroperoxide (TA102). With S9: 2-Amino-anthracene (TA97a, TA98, TA100, TA1535), 1,8-Dihydroxy-anthraquinone (TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: not reported

OTHER EXAMINATIONS:
- Determination of polyploidy: not done
- Determination of endoreplication: not done

Evaluation criteria:

The criteria for a positive result are a reproducible statistically significant increase of the number of revertants to more than twice the amount of the spontaneous revertants for at least one of the concentrations.

Statistics:

For comparison of the control group and the test substance groups the analysis of variance was used followed by the test of Scheffe. For comparison of the control group and the positive control group, if the results were not clear beyond any doubt, the t-test was used. p = 0.05 was used as the significance level.

Results and discussion

Test results

Species / strain:

other: TA97a, TA98, TA100, TA 102 and TA1535

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: As in the preliminary test no toxicity was obtained, no attempt was made to reduce the high pH. The petri dishes after the incubation had a pH of about 7, so the buffer in the bacterial media was strong enough to reduce the alkalinity of the test substance to allow bacterial growth.
- Water solubility: The test substance was easily soluble in water in the required concentration for the stock solution (50 mg/mL).
- Precipitation: No visible precipitation occurred during the test.

RANGE-FINDING/SCREENING STUDIES: The test substance was not toxic to strain TA100 up to 5000 µg per plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

According to the results obtained in this study, Guanidine carbonate is non-mutagenic in the Ames test with the strains TA97a, TA98, TA1I 00, TA102 and TA1535 up to 5000 µg per plate which is the limit concentration for this test.

Executive summary:

Guanidine carbonate was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD TG 471.

The test substance, dissolved in water, was tested at concentrations ranging from 62 µg to 5000 µg per plate according to the "direct plate incorporation method" without external metabolization as well as with an external metabolising system (S9-mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed.

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

The test substance was easily soluble in water, no visible precipitation occurred during the test. The test substance was not toxic up to the concentration of 5000 µg/plate which is the limit concentration for this kind of test.

In none of the concentrations tested and with none of the strains used a statistically significant increase of the mutation frequency to more than twice the amount of the control samples was obtained. Metabolic activation did not change these results.

According to the results obtained in this study, Guanidine carbonate is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg per plate which is the limit concentration for this test.