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Genetic toxicity in vivo

Description of key information
The results from one Ames in vitro bacterial study, two in vitro chromosomal aberration studies (one with cultured human lymphocytes and one with Chinese hamster fibroblasts) and one in vivo Mammalian Erythrocyte Micronucleus Test in mice are available. All tests were negative for genotoxicity, except the chromosomal aberration study with cultured human lymphocytes which was positive in absence of metabolic activating system.
Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
as at 1997
Deviations:
no
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
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
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
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
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
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)

- 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

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.

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

Additional information

Additional information from genetic toxicity in vivo:

A total of four guideline or equivalent studies have been conducted with Guanidine carbonate. In a key study, the test substance was negative for mutagenicity when tested at plate incorporation levels of up to 5000 micrograms/plate with Salmonella sp. TA97a, TA98, TA100, TA 102 and TA1535, either with or without rat liver S9 -mix.

In a key study with cultured human lymphocytes, the subject chemical induced a clear statistically significant increase in the numbers of metaphases with structural aberrations at the low concentration of 0.56 mg Guanidine carbonate per mL medium after 3 hours of test substance treatment and at the highest analysable concentration of 1.67 mg/mL after 20 hours of treatment. This response was not repeated in the chromosome aberration test with Chinese hamster fibroblasts. However, in the in vivo MNT in mice no clastogenic effects could be observed. Therefore, the response seen in cultured human lymphocyte cells was considered of no toxicological significance.


Justification for selection of genetic toxicity endpoint
Three in vitro test and one in vivo test on genotoxicity are available. The in vivo test overrules the in vitro tests and is therefore selected.

Justification for classification or non-classification

Guanidine carbonate was negative when tested in vitro in the Ames bacterial mutagenicity assay. In one in vitro chromosomal aberration study the test substance induced chromosomal aberration in absence of metabolic activating system. However, in the in vivo MNT no clastogenic effects with Guanidine carbonate could be observed.

Based on the results of these in vitro and in vivo studies, the substance does not need to be classified according to CLP (Regulation (EC) No 1272/2008).

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