Diguanidinium carbonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: - OECD guideline compliant - GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

as at 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

as in Directive 2000/32/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

593-85-5

IUPAC Name:

593-85-5

Constituent 2

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

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Mice, Crl:NMRI BR
- Source: Charles River WIGA GmbH, D-97633 Sulzfeld
- Age at study initiation: 9 wks
- Weight at study initiation: males: 34.04 ± 2.0 g, females: 27.53 ± 1.5 g
- Assigned to test groups randomly: yes
- Fasting period before study: no, overnight fasting before administration
- Housing: males: single caging, Makrolon cages type II (22 cm x 16.5 cm x 14 cm), wire mesh lids; females: five animals per cage, Makrolon cages, type III, low version (39 cm x 23 cm bottom area, 15 cm height), wire mesh lids; Aspen wood chips, type "4 HV" (Finn Tapvei Ky, SF-73600 Kaavi), germ reduction by autoclaving, changed once a week
- Diet (e.g. ad libitum): ad libitum, Altromin standard diet for rats and mice, No. 1324 forte, germ reduction by 25 kGy 60Co gamma irradiation
- Water (e.g. ad libitum): ad libitum, tap water, offered in Makrolon bottles with stainless steel canules
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 46.2
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The test item was known to be sufficiently water soluble
- Concentration of test material in vehicle: 40, 80, 120 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- substance dissolved in deionised water

Duration of treatment / exposure:

single oral treatment

Frequency of treatment:

single oral treatment

Post exposure period:

animals sacrificed 24 or 48 h post dosing for tissue sampling

Doses / concentrations

Remarks:

Doses / Concentrations:
400, 800 and 1200 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide
- Justification for choice of positive control(s): recommended in the OECD TG 474
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow cells obtained from both femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- range-finding study, doses of 500, 1000 and 1500 mg
- One male and one female at 1500 mg/kg bw died on the day of the test substance administration; all other animals survived until 48 hours post dosing
- no marked cytotoxicity was noted at the evaluation of the bone marrow at any dose
- dose of 1200 mg Guanidine carbonate chosen as high dose for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- treatment: single oral gavage
- sampling times: 24 and 48 h post dosing

DETAILS OF SLIDE PREPARATION:
- animals killed by cervical dislocation
- bone marrow obtained from both femurs according to the method of W. SCHMID (The micronucleus test for cytogenetic analysis. In: Hollaender, Chemical Mutagens, Vol.4; Plenum Press New York and London, 1976).
- three smears per animal
- two smears stained (slightly modified Pappenheim method) coded and scored, decoding after scoring of last slide

METHOD OF ANALYSIS:
- Leitz DMRB microscope at a magnification of about 630 or 1000
- determination of the ratio of nucleated cells: >= 200 cells/slide analysed
- determination of the ratio of polychromatic to normochromatic erythrocytes: >= 500 erythrocytes per slide analysed
- determination of rate of micronucleated erythrocytes: 1000 cells per slide analysed (2000 per animal)

Evaluation criteria:

Not reported in detail, but the reporting of the results indicates that the criteria described in OECD TG 474 were used.

Statistics:

- Micronucleated cells:
U-test of Wilcoxon, Mann and Witney: for comparison of two groups
H-test of Kruskal and Wallis followed by the test of Nemenyi: for comparison of more than two groups

- Body weights, composition of bone marrow:
t-test: for comparison of two groups
Analysis of variance followed by the Scheffe test: for comparison of more than two groups

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000, 1500 mg/kg bw
- Solubility: soluble at all doses
- Clinical signs of toxicity in test animals: one male and one female at 1500 mg/kg bw died on the day of the test substance administration; all other animals survived until 48 hours post dosing
- Evidence of cytotoxicity in tissue analyzed: no marked cytotoxicity was noted at the evaluation of the bone marrow at any dose
- Harvest times: 48 h

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
• 24 hours p.a., rise in the amount of micronucleated polychromatic erythrocytes in one female dosed with 800 mg per kg body weight and in one female dosed with 1200 mg/kg as compared to historical negative controls. No statistically significant differences in the amounts of micronucleated polychromatic erythrocytes compared to the corresponding negative control groups, neither 24 nor 48 hours p.a.
- Ratio of PCE/NCE (for Micronucleus assay):
• No marked differences in the amounts of nucleated cells and of polychromatic erythrocytes between the test substance groups and the corresponding negative controls.
- Appropriateness of dose levels and route: Dose levels evaluated in a pretest and based on systemic toxicity (deaths)

Any other information on results incl. tables

- Table 1: Summarized results

group (dose)	Doses (mg/kg bw)	Sampling time (h post dosing)	parameter	NC %	PE %	NE %	ratio PE/NE	MPE (1/1000)	MNE (1/1000)
Females
NC 1 negative control	-	24	mean	70.2	67.2	32.8	2.10	0.50	0
			sd	8.2	4.5	4.5	0.51	0.71	0
			n	5	5	5	5	5	5
NC 2 negative control	-	48	mean	63.6	63.9	36.1	1.88	0.30	0.51
			sd	9.6	7.1	7.1	0.68	0.27	1.15
			n	5	5	5	5	5	5
A test substance	400	24	mean	65.5	67.5	32.5	2.15	0.50	1.13
			sd	11.0	6.3	6.2	0.48	0.87	1.59
			n	5	5	5	5	5	5
B test substance	800	24	mean	63.8	66.6	33.4	2.09	0.80	0
			sd	6.6	6.3	6.3	0.64	1.52	0
			n	5	5	5	5	5	5
C1 test substance	1200	24	mean	70.2	66.1	33.9	1.98	2.00	2.75
			sd	6.6	3.7	3.7	0.35	2.15	3.34
			n	5	5	5	5	5	5
C2 test substance	1200	48	mean	62.9	66.7	33.3	2.04	0.20	0.68
			sd	8.3	4.2	4.2	0.39	0.45	1.53
			n	5	5	5	5	5	5
PC positive control	40	24	mean	51.9	57.3	42.7	1.54	7.00	0
			sd	9.7	13.1	13.1	0.83	3.74	0
			n	5	5	5	5	5	5
Males
NC 1 negative control	-	24	mean	62.1	63.2	36.8	1.81	0.70	0.50
			sd	8.2	7.5	7.5	0.60	0.76	1.12
			n	5	5	5	5	5	5
NC 2 negative control	-	48	mean	62.0	68.61	31.4	2.25	0.80	0.55
			sd	3.4	5.1	5.1	0.55	0.84	1.24
			n	5	5	5	5	5	5
A test substance	400	24	mean	72.1	69.8	30.2	2.38	1.00	0.56
			sd	4.9	4.5	4.5	0.51	0.61	1.24
			n	5	5	5	5	5	5
B test substance	800	24	mean	66.4	71.1	28.9	2.50	0.60	0
			sd	5.6	3.6	3.6	0.43	0.55	0
			n	5	5	5	5	5	5
C1 test substance	1200	24	mean	71.0	68.1	31.9	2.31	1.20	0
			sd	2.7	7.3	7.3	0.95	0.57	0
			n	5	5	5	5	5	5
C2 test substance	1200	48	mean	70.5	63.9	36.1	1.80	0.20	0
			sd	3.3	4.1	4.1	0.32	0.27	0
			n	5	5	5	5	5	5
PC positive control	40	24	mean	47.5	50.8	49.2	1.06	15.60	1.69
			sd	12.4	6.9	6.9	0.30	8.26	2.38
			n	5	5	5	5	5	5

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Guanidine carbonate was tested in a Mammalian Erythrocyte Micronucleus Test conducted according to OECD Guideline 474 and GLP. Under the test conditions described, Guanidine carbonate did not produce relevant increases of the numbers of micronuclei in polychromatic erythrocytes in animals of either sex of the test species at doses of 400, 800 or 1200 mg/kg body weight and sampling times of 24 and 48 hours p.a.

Executive summary:

Guanidine carbonate was tested in a Mammalian Erythrocyte Micronucleus Test conducted according to OECD Guideline 474 and GLP. Due to the results obtained in this study and under the test conditions described guanidine carbonate did not produce relevant increases of the numbers of micronuclei in polychromatic erythrocytes in animals of either sex of the test species at doses of 400, 800 or 1200 mg/kg body weight and sampling times of 24 and 48 hours p.a.

Guanidine carbonate, dissolved in deionised water, was administered once orally by gavage at doses of 400, 800 or 1200 mg per kg body weight to groups of 5 male and 5 female Crl:NMRI BR-mice each. Two negative control groups (deionised water) and one positive control group (cyclophosphamide, dissolved in deionised water) were also included in the study. The dose volume was 10 mL per kg body weight for all groups.

Preparation of bone marrow cells and investigations were performed in conformance with the Directive 2000/32/EC, B.12 and with the OECD Guideline 474 (1997).

All animals survived until the scheduled sacrifice. No test substance related adverse effects were noted in life. In the range finding study mortality occurred at a dose of 1500 mg per kg body weight.

No cytotoxic effects of the test substance were noted, neither in males nor in females, neither 24 nor 48 hours p.a.

There were no significant differences in micronucleated normochromatic erythrocytes between the test substance group animals of both sexes and the corresponding negative controls, neither 24 nor 48 hours after administration. However, the parameter of major interest is the amount of micronucleated polychromatic erythrocytes. In this test system, a test substance is considered to induce chromosomal damage or damage to the mitotic apparatus, if a significantly increased amount of micronucleated polychromatic erythrocytes compared to the corresponding negative controls is detected. 24 hours p.a., the amount of micronucleated polychromatic erythrocytes was higher in one female dosed with 800 mg per kg body weight and in one female dosed with 1200 mg/kg than found in historical negative controls. However, statistical analysis did not show significant differences in the amounts of micronucleated polychromatic erythrocytes compared to the corresponding negative control groups, neither 24 nor 48 hours p.a. All mean data were also within the range of historical negative controls.

Cyclophosphamide caused cytotoxicity and produced micronuclei in polychromatic erythrocytes, thus demonstrating the sensitivity of the test system used for the endpoints investigated in this study.

No marked differences between the sexes were noted in the response to the test substance.