1-nitroguanidine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of nitroguanidine was assessed in several in vitro tests and one in vivo assay. In vitro gene mutation in bacteria (Ames test) was negative, both in the presence and the absence of metabolic activation. Two in vitro cytogenicity studies in mammalian cells are available showing negative results. The potential to induce sister chromatid exchanges (SCEs) was shown to be negative using Chinese hamster ovary cells (CHO) both with and without exogenous metabolic activation. Nitroguanidine was did not induce structural chromosomal aberrations in human lymphocytes in a recent guideline-compliant study (OECD473). In a rapid in vitro screening test in CHO cells without metabolic activation chromsome aberrations were detected, but this published study was found to be poorly conducted, not fulfilling guideline requirements, and therefore to be invalid. In vitro gene mutation in mammalian cells was shown to be absent by means of a mouse lymphoma forward mutation assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 2013 - 02 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Test substance supplier NIgu Chemie GmbH
Los 3286

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

- collected from a single donor who was a healthy, non-smoking individual with no kown recent exposures to pharmaceuticals, genotoxic chemicals, or radiation
- drawn by venous puncture and collected in heparinized tubes
- blood was stored under sterile conditions at 4 derees C for a maximium of 4 hours
- whole blood samples treated with an anti-coagulant were pre-cultured in the presence of mitogen

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

without: EMS; with: CPA

Test concentrations with justification for top dose:

-Experiment 1: without metabolic activation - 15, 20, 180, 360, 750, 1250, 2500, 3000, 4000, 5000 ug/mL
-Experiment 1: with metabolic activation - 180, 360, 750, 1250, 2500, 3000, 3500, 4000, 5000 ug/mL
-Experiment 2: without metabolic activation - 750, 1250, 2500, 3000, 3500, 4000, 4500, 5000 ug/mL
-Experiment 2: with metabolic activation - 500, 1000, 2000, 3500, 4500, 5000 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI + 0 % fetal bovine serum
- Justification for choice of solvent/vehicle: based on the results of the solubility test cell culture medium cell culture medium was used as a solvent

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Experiment 1: without and with metabolic activation, 4h treatment, 24h preparation interval - 1250, 2500, 5000 ug/mL
- Experiment 2: without metabolic activation, 24h treatment, 24h preparation interval - 1250, 2500, 5000 ug/mL
- Experiment 2: with metabolic activation, 4h treatment, 24h preparation interval - 1000, 2000, 5000 ug/mL
- at least 3 analysable concentratiosn of hte test item were used for the 24 h preparation

DETERMINATION OF CYTOTOXICITY
- Method: proliferation index, mitotic index

Evaluation criteria:

The chromosomal aberration assay is considered acceptable if:
- The number of aberration found in the negative and/or solvent controls fall within the range of historical laboratory control data: 0-4 % (without and with metabolic activation)
- The positive control substance should produce biologically relevant increases in the number of cells with structural chromosome aberrations

Criteria for determining a positive result:
- A clear and dose-related increase in the number of cells with aberrations
- A biologically relevant response for at least one dose group, which is higher than the laboratory negative control range (0-4 % without and with metabolic activation)

Statistics:

- Statistical significance (P < 0.05) was determined by Fischer's exact test

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The test substance precipitates at 2 and 5 mg, therefore there is no genotoxicity up to the maximum solubility.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment 2, with and without metabolic activation, a biologically relevant decrease of the relative mitotic index was seen at 5000 ug/mL.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Nitroguanidine did not induce structural chromosomal aberrations in human lymphocytes and is therefore considered non-clastogenic. Furthermore, the test substance precipitates at 2 and 5 mg, therefore there is no genotoxicity up to the maximum solubility.

Executive summary:

In a mammalian cell cytogenetics chromosomal aberration assay primary human lymphocyte cultures were exposed to nitroguanidine at the following concentrations:

-Experiment 1: without metabolic activation - 15, 20, 180, 360, 750, 1250, 2500, 3000, 4000, 5000 µg/mL

-Experiment 1: with metabolic activation - 180, 360, 750, 1250, 2500, 3000, 3500, 4000, 5000 µg/mL

-Experiment 2: without metabolic activation - 750, 1250, 2500, 3000, 3500, 4000, 4500, 5000 µg/mL

-Experiment 2: with metabolic activation - 500, 1000, 2000, 3500, 4500, 5000 µg/mL 

Nitroguanidine was tested up to 5000 µg/mL because the guideline recommends a maximum concentration of 5 mg/mL. Positive controls induced the appropriate response. There was no evidence of chromosomal aberration induced over background.

This study is classified as acceptable.  This study satisfies the requirement of test guidelines OECD 473 (in vitro mammalian chromosome aberration test), EU method B.10 (mutagenicity - in vitro mammalian chromosome aberration test), and EPA OPPTS 870.5375 - in vitro mammalian chromosome aberration test for in vitro cytogenetic mutagenicity data.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: LAIR SOP op-STX-72 1985

Deviations:

yes

Remarks:

during harvesting phase: instead of shaking each rinsed flask for 10 min with HBSS, the flasks were shaken for 30 min. This provided sufficient time for the cells still adhering to the flask to detach.

GLP compliance:

yes

Remarks:

GLP Study 85036

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

Test substance supplier Sunflower AAP
Assey 99.2%

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cells were grown in an atmosphere of 5% CO2 at 37 +/- 1 °C in GEM 1717 with 10% FBS
- Cell cycle time: 10-12 h

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

- Without metabolic activation: 4 mg/ml to 0.01 mg/ml
- With metabolic activation: 3.9 mg/ml to 0.01 mg/ml

Vehicle / solvent:

Nitroguanidine was dissolved directly in the cell culture medium (GEM 1717)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cultures without metabolic activation: ethyl methanesulfonate (EMS), final concentration 0.320 mg/ml; cultures with metabolic activation: cyclophosphamide, final concentration 1.33 mg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Nitroguanidine was dissolved directly in cell culture medium (GEM 1717), without fetal bovine serum (FBS), at a conc. of 4.4 mg/ml and was filter-sterilized -> stock solution
- For the non activated cultures, FBS was added to the culture medium to achieve a final concentration of 10 %
- Dilutions were prepared using GEM 1717 with 10% FBS
- For cultures with metabolic activation, two batches of cofactor mixture were prepared; one containing nitroguanidine while the other did not, and nitroguanidine dilutions were prepared by mixing the two solutions


MEDIUM:
- HEPES buffered (10nM) GEM 1717
- Prepared from RPMI 1640
- Contains 10% (v/v) FBS when in contact with cells except when the medium was used to prepare the activation cofactor mixture

ASSAY FORMAT:
- Test without metabolic activation:
* Test compound was added to fresh GEM 1717 with 10% FBS (15 ml per flask) containing 5 µM BrdU
* Cells were then grown in the dark at 37 +/- 1 °C for 21 h
* Flasks were wrapped in foil to assure complete darkness
* Then colchicine was added to each flask (0.4 µg/ml)
* The cultures were incubated for an additional 2.5 h before the cells were harvested
- Test with metabolic activation:
* Complete medium was removed and 15 ml of activation mixture, with or without the test article or positive control, was added
* Cell cultures were exposed to the test compound in the activation mixture for 2 hours (shorter exposure duration to preclude the occurrence of cytotoxic effects induced by activation mixture)
* After 2 h exposure period, the medium containing both the test compound and the metabolic activation mixture was aspirated, the cells were washed 3 x with GEM 1717, and fresh, complete GEM 1717 containing 190% FBS and 5 µM BrdU was added
* The cultures were incubated in the dark for 21.5 h
* Colchicine was then added at a final concentration of 0.4 µg/ml as described above and the cultures were incuated for an additional 2.5 h before the cells were harvested

HARVEST:
- After 2.5 h in colchicine, all of the cells in each culture were harvested
- Medium containing dividing cells was removed from the culture flasks and saved
- Each flask was rinsed once with 10 ml of Ca2+ Mg2+ free Hank's Balanced Salt Solution (HBSS)
- This wash was combined with the culture medium in a 50 ml tube
- 10 ml of Ca2+ Mg2+ free HBSS (with 0.02 % EDTA) were added to the rinsed flasks, which were then shaken for approx. 30 min, until almost all the remaining cells were detached
- Contents of 50ml tubes were spun down ( 1000 rpm for 5 min) and all but 3 ml of the supernatant above each cell pellet was discarded
- Each cell pellet was resuspended in its 3 ml of supernatant, and these cell suspensions were transferred to prelabelled 15-ml centrifuge tubes
- Cell suspension of from each flask was added to the appropriate tube and cells were centrifuged ( 1000 rpm for 5 min)
- Most of the supernatant was aspirated, and each cell pellet was gently resuspended in the remaining supernatant (approx. 0.2 to 0.3 ml/tube)
- 10 ml of hypotonic KCl (0.075 M) were added slowly to each tube, and the tubes were held at room temperature for approx. 2 min
- Tubes were again centrifuged (1000 rpm for 5 min), the supernatant aspirated, and the cell pellet gently resuspended in the approx. 10.3 ml of remaining supernatant
- Carnoy's fixative was then added to each tube
- Overnight fixation
- Cells were then centrifuged and fixation procedure was repeated
- Cells were then centrifuged and all but about 0.5 ml supernatant was removed, and the pellet was resuspended
- Prelabelled clean slides were wetted in cold distilled water; up to 4 drops of cell suspension were dropped onto each slide


STAINING:
- Slides were stained by a modified FPG technique:
* Several drops of Hoechst 33258 (50 µg/ml in water) were placed on each slide, and a coverslip was applied
* After standing in the dark for 15 min, the coverslips were removed and the slides were washed several times in tap water and allowed to dry.
* Several drops of McIlvaine's buffer (pH 8) were placed on each slide, and a new coverslip was applied
* Slides were placed on the 50 +/- 2 °C warming table under a UV lamp
* The slides were exposed to the UV light to induce photocleavage of the DNA strands
* The coverslips were removed, and the slides were rinsed several times in tap water and dried; then they were stained in dilute Giemsa stain (5 % Gurr's Giemsa pH 7.0), rinsed, and air dried; then a coverslip was applied


SCORING:
- For each dose group triplicate slides were prepared and coded
- Number of first-, second-, and third-division metaphases were scored (where possible, 100 metaphases were scored)
- Normally, 40 second-division metaphases, containing 20 chromosomes, were scored for SCEs in each preparation
- In some cases there were less than 40 second-division metaphases to score or the number of SCEs per cell was sufficiently high so that fewer cells needed to be counted to achieve statistical significance
- In the first case, all available metaphases (with 20 chromosomes) were scored on all slides


DURATION
- 24 h before treatment, 6 x 10^5 cells were seeded into each 75 cm^3 flask, which contained 15 ml of fresh medium, to ensure that the cells were in the logarithmic phase of growth when the test compound was added
- Exposure duration: without metabolic activation: 24 hours; with metabolic activation: 2 hours



SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS: triplicate


NUMBER OF CELLS EVALUATED:
where possible, 100 metaphases were scored

Evaluation criteria:

Validity criteria:
A valid SCE assay is one in which (1) testing was performed to the maximum concentration expected to yield sufficient second-division metaphases for analysis, the limits of solubility, or the limits of compound availability (a maximum of 5000 µg/ml or 0.5 % for liquids), and (2) the positive control induced a statistically significant increase in the SCE frequency.

Interpretation of results:
- Positive: A test compound will be considerd to have elicited a positive response in the SCE assay if it induces a statistically significant (p < 0.05) increase in the SCE frequencies compared to the negative control rate (by the Student-Newman-Keuls test) and the SCE frequency exhibited a correlated dose response
- Negative: A test compound will be considerd to have elicited a negative response in the SCE assay if the criteria (statistical significance or dose response) for a positive response are not met.

Statistics:

Statistical evaluation of the SCEs data was a two-step process involving the use of a one-way analysis of variance to compare variance within treatments (due to scoring difference and sample difference) with variance among treatments (the effect of test compound) followed by a Student-Newman-Keuls test to indicate which groups (treatments) were different. Thus, the ANOVA was used to indicate a difference among groups and the Student-Newman-Keusl test was used to indicate which specific dose or group of doses was different from each other or controls. The positive and negative controls were compared by using the t-test. The p < 0.05 confidence limit was used for the t-test, ANOVA, and Student-Newman-Keuls test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see remarks on results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Positive controls induced significant increase in SCEs.

At 4.0 mg/ml (without metabolic activation), 91% of the metaphases isolated were found to be from first-division cells, and the total yield of metaphases was to small for SCR scoring. The 2.0 mg/ml (without metabolic activation) dose indiced some toxicity during the 24 hour exposure but failed to induce a significant raise in SCEs. None of the lower doses of nitriguanidine induced toxicity or increase in SCEs.

Table 1: Nitroguanidine (NG) Sister Chromatide Exchange

Doses		% FDA^a	n^b	SCE	± 1 SD	Comment
Without S-9 (24-hour exposure)
Neg control		2	40	6.58	± 2.86
EMS	0.32 mg/ml	12	6	61.00	± 9.74	p < 0.0001
NG	4.0 mg/ml	91	40	metaphases not scorable
NG	2.0 mg/ml	14	40	7.28	± 2.53	NS
NG	1.0 mg/ml	3	40	7.30	± 2.66	NS
NG	0.5 mg/ml	2	40	7.20	± 3.03	NS
NG	0.1 mg/ml	1	40	6.78	± 2.83	NS
NG	0.05 mg/ml	7	40	6.25	± 2.26	NS
NG	0.01 mg/ml	4	40	7.25	± 2.65	NS
					±
With S-9 (2-hour exposure)
Neg control		1	39	9.31	± 2.57
Cyclophosphamide	1.33 µg/ml	9	20	34.5	± 11.75	p < 0.0001
NG	3.9 mg/ml	1	39	9.03	± 3.27	NS
NG	2.0 mg/ml	1	19	6.53	± 2.57	NS
NG	1.0 mg/ml	2	28	8.00	± 3.10	NS
NG	0.5 mg/ml	1	40	7.85	± 3.31	NS
NG	0.1 mg/ml	4	20	8.60	± 3.22	NS
NG	0.05 mg/ml	0	40	8.65	± 3.32	NS
NG	0.01 mg/ml	4	40	7.00	± 2.66	NS

^aFDA = percentage of metaphases in first division

^bn 0 number of metaphases scored for SCEs

Negative controls and nitroguanidine groups were compared using ANOVA.

Negative controls and positive controls were compared using the t-test.

Abbreviations: EMS = ethyl methanesulfonate, NG = nitroguanidine, SCE = sister chromatide exchange, SD = standard deviation, NS = not significant (p ≤ 0.05)

Conclusions:

The results indicate that nitroguanidine was not an inducer of SCEs under the conditions of this study.

Executive summary:

The potential of nitroguanidine to induce Sister Chromatid Exchanges (SCEs) was assessed using Chinese Hamster Ovary (CHO) cells, both with and without exogenous metabolic activation provided by rat liver S-9.

Cells were exposed to test compound concentrations ranging from 4 mg/ml to 0.01 mg/ml in cultures without exogenous metabolic activation and 3.9 mg/ml to 0.01 mg/ml in cultures with exogenous activation.

Nitroguanidine did not induce a statistically significant increase in SCEs in either the presence or absence of exogenous metabolic activation.

These results indicate that nitroguanidine was not an inducer of SCEs under the conditions of this study.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

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:

a revision of the Ames Salmonella/Mammalian Microsome Assay was used (Maron; Ames. Mutat Res 1983; 113: 173-215

Deviations:

yes

Remarks:

(Volumes for preincubation different from those specified in method because of limited solubility of nitroguanidine)

Principles of method if other than guideline:

Two new test strains, a frame-shift strain and a strain carrying an ochre mutation on a multicopy plasmid (TA102), were added to the standard test set.

GLP compliance:

yes

Remarks:

GLP study number 86008

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

Target gene:

- genes involved in histidine synthesis

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA102, TA1535, TA1537, and 1538

Additional strain / cell type characteristics:

other: histidine auxotrophy

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

- highest concentration: 2.8 mg/ml
- serial dilution
- lowest concentration: 0.0875 mg/ml

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in grade I DMSO

Positive control substance:

other: benzo(a)pyrene, 2-aminofluorene, 2-aminoanthracene, mitomycin-C, 4-nitroquinoline-n-oxide, N-methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation


DURATION
- Preincubation period: 20 min at 37 °C on a shaker incubator


NUMBER OF REPLICATIONS:
triplicates

Evaluation criteria:

According to Brusick, a compound is considered mutagenic if a positive dose response (correlated dose response) over three dose concentrations is achieved with at least the highest dose yielding a revertant colony count greater than or equal to twice the spontaneous colony count for the tester strains TA98 and TA100, or three times the spontaneous colony count for strains TA1535, TA1537, and TA1538. A strong correlated dose response in strain TA100 without doubling of the individual colony count may also be considered positive.

Maron and Ames consider a compound mutagenic in tester strains TA97 and TA102 if a correlated dose response over three concentrations is achieved with the highest dose yielding a revertant colony count greater than or equal to twice the spontaneous colony count.

Statistics:

- Spontaneous reversion rate was compared with historical values

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

STERILITY AND STRAIN VERIFICATION:
- Normal results were obtained for all tests

Nitroguanidine did not induce any appreciable increase in the revertant colony counts relative to those of the negative control cultures

Table 1: Nitroguanidine Revertants/Plate Mean± 1SD

Compound*	Dose/ plate	TA97	TA98	TA100	TA102	Compound*	Dose/ plate	TA1535	TA1537	TA1538
WITHOUT S-9
Neg. control	0.00 mg	88 ± 16.1	14 ± 3.3	111 ± 10.2	173 ± 13.2	Neg. control	0.00 mg	11 ± 3.0	10 ± 3.2	18 ± 6.4
Mito C	0.05 µg				1327 ± 22.5	MNNG	20.0 µg	2224 ± 378.2
MNNG	2.0 µg					TP 36A	2.8 mg	21 ± 2.5	19 ± 4.6	19 ± 4.0
MNNG	20.0 µg			1460 ± 149.1		TP 36A	1.4 mg	13 ± 4.7	7 ± 0.6	10 ± 4.6
NQNO	2.0 µg	252 ± 92.2				TP 36A	0.7 mg	10 ± 2.3	12 ± 5.6	9 ± 3.6
TP 36A	2.8 mg	66 ± 14.6	19 ± 0.6	88 ± 22.5	119 ± 1.7	TP 36A	0.35 mg	10 ± 1.2	10 ± 0.0	16 ± 5.9
TP 36A	1.4 mg	96 ± 14.6	13 ± 3.8	107 ± 2.9	198 ± 2.0	TP 36A	0.0175 mg	14 ± 4.0	6 ± 2.0	13 ± 0.6
TP 36A	0.7 mg	83 ± 15.3	12 ± 4.6	106 ± 11.0	166 ± 24.4	TP 36A	0.0875 mg	16 ± 6.4	5 ± 0.6	17 ± 1.0
TP 36A	0.35 mg	102 ± 27.7	12 ± 3.2	85 ± 15.5	162 ± 15.0
TP 36A	0.0175 mg	96 ± 7.6	20 ± 2.0	96 ± --	146 ± 35.6
TP 36A	0.0875 mg	93 ± 6.1	16 ± 5.5	99 ± 13.1	172 ± 12.7
WITH S-9
Neg. control	0.0 mg	110 ± 16.6	35 ± 10.8	125 ± 21.7	282 ± 5.4	Neg. control	0.00 mg	11 ± 1.7	12 ± 4.1	27 ±2.4
2-AA	2.0 µg		298 ± 73.4	563 ± 86.5		2-AA	2.0 µg		318 ± 55.2	572 ± 231.1
2-AF	2.0 µg	572 ± 136.7	1683 ± 42.5	706 ± 86.5		2-AF	2.0 µg			1168 ± 173.9
BP	2.0 µg	251 ± 7.5	125 ± 7.5	366 ± 35.8		BP	2.0 µg		48 ± 5.9	74 ± 39.0
TP 36A	2.8 mg	144 ± 38.3	38 ± 8.5	110 ± 36.2	272 ± 27.2	TP 36A	2.8 mg	9 ± 3.6	10 ± 3.6	25 ± 9.6
TP 36A	1.4 mg	107 ± 9.5	25 ± 2.1	122 ± 26.6	282 ± 20.4	TP 36A	1.4 mg	18 ± 13.0	10 ± 4.2	34 ± 1.7
TP 36A	0.7 mg	121 ± 7.6	30 ± 1.7	99 ± 6.4	308 ± 13.9	TP 36A	0.7 mg	12 ± 4.9	9 ± 2.5	25 ± 4.9
TP 36A	0.35 mg	101 ± 9.0	26 ± 1.0	127 ± 22.7	280 ± 10.2	TP 36A	0.35 mg	14 ± 4.5	11 ± 4.2	14 ± 6.5
TP 36A	0.0175 mg	115 ± 4.6	27 ± 0.6	105 ± --	267 ± 59.5	TP 36A	0.0175 mg	15 ± 3.0	12 ± 4.6	24 ± 7.0
TP 36A	0.0875 mg	107 ± 6.6	33 ± 4.5	129 ± 9.7	298 ± 15.1	TP 36A	0.0875 mg	16 ± 11.8	12 ± 2.5	17 ± 1.0

* MITO-C = mitomycin-C; MNNG = N-methyl-N’-nitro-N-nitrosoguanidine; NQNO = 4-nitroquinoline-n-oxide; AA = 2-aminoanthracene, AF = 2-aminofluorene; BP = benzo(a)pyrene; TP 36A = test substance nitroguanidine

Conclusions:

Nitroguanidine was evaluated for mutagenic potential in the Ames test, both in the presence and the absence of metabolic activation, and did not induce a positive mutagenic response under the conditions of this study.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA97, TA98, TA102, TA1535, TA1537, and 1538 of S. typhimurium were exposed to Nitroguanidine. The test was performed with and without the addition of S-9 mix as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at a concentration range from 2.8 mg/plate to 0.0875 mg/plate in the presence and absence of a metabolic activation system. The experiment was conducted in triplicates.

The experiments with and without metabolic activation were performed as preincubation assay.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of Nitroguanidine induced a positive mutagenic response under the conditions of this study.

 

Based on the results of these experiments, it is concluded that Nitroguanidine did not induce gene mutations in the strains of S. typhimurium used.

This study is classified as acceptable. This study satisfies the requirement for the revised Ames Salmonella/Mammalian Microsome Assay.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1980

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

- E. coli DNA Repair Test on Plates
- S. typhimurium Mutation Test
- S. cerevisiae Mitotic Recombinogenic Activity Test

> no details reported

GLP compliance:

not specified

Type of assay:

other: DNA repair assay; reverse mutation assay; mitotoc recombinogenic test

Specific details on test material used for the study:

Test substance supplier not specified

Species / strain / cell type:

E. coli, other: W3110/polA+; p3478/polA-

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Species / strain / cell type:

Saccharomyces cerevisiae

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

no data

Vehicle / solvent:

no data

Details on test system and experimental conditions:

no data

Evaluation criteria:

no data

Statistics:

no data

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

E. coli, other: W3110/polA+; p3478/polA-

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

Saccharomyces cerevisiae

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Nitroguanidine was not genotoxic in any of the test systems.

Executive summary:

Nitroguanidine was subjected to DNA repair assays using the E. coli W3110/polA+, p3478/polA- system, reverse mutation assays with His- Salmonella typhimurium strains TA2535, TA1537, TA1538, TA98, and TA100, and mitotoc recombinogenic tests with the yeast Saccharomyces cerevisiae D5. Tests were carried out in the absence of an exogenous activation system and in tissue homogenate-mediated assays using Aroclor 1254 -induced, male rat-liver-derived S9 mix.

Nitroguanidine was not genotoxic in any of the test systems.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: LAIR SOP, OP-STX-71

Deviations:

yes

Remarks:

In the initial assay, the conc. of 2AAF was 0.5 mg/ml instead of 0.05 mg/ml, which accounts for the extreme toxicity observed. This toxicity and the mutant frequency does, however, demonstrate that the activation system was functioning.

GLP compliance:

yes

Remarks:

GLP Study Number: 85035

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Test substance supplier Sunflower AAP

Target gene:

gene for thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: cells were maintained in antibiotic-free Fisher's Medium for Leukemic Cells of Mice (Fisher's Medium) supplemented with 10% horse serum
- Six days before each assay began, the cell population was cleared of spontaneous thymidine kinase negative mutants by methotrexate treatment and screened for mycoplasma and other contaminants by using the 3T6 co-culture technique
- No nonnuclear DNA was detected after four days of co-culture, and thus the cell line was presumed to be uncontaminated

Additional strain / cell type characteristics:

other: 3.7.2C TK+/-

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

Assay without metabolic activation: 4, 2, 1, 0.5, 0.1, 0.05, and 0.01 mg/ml
Assay with metabolic activation: 3.66, 2, 1, 0.5, 0.1, 0.05, and 0.01 mg/ml

Vehicle / solvent:

- Vehicle used for positive control with metabolic activation: DMSO
- The final DMSO concentration of the 2AAF treated cultures did not exceed 1 %

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethyl methanesulfonate (EMS): 0.32 mg/ml in assay without metabolic activation; 2-acetamide fluorene (2AAF) in DMSO: 0.5 mg/ml (first assay) or 0.05 mg/ml (confirmatory assay) in assays with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Nitroguanidine was dissolved directly in Fisher's Medium without serum with a final concentration of 4 mg/ml
- 4 mg/ml is close to saturation for nitroguanidine in an aqueous medium
- Medium: immediately filter sterilized after preparation and then supplemented with 1-glutamine (2 mM) and sodium pyruvaze (1 mM)
- Sterile horse serum was heat inactivated before use (56 °C for 30 min)
- Fisher's medium was supplemented with horse serum at 5 %, 10 %, or 20 % (v/v) final concentration -> designated F5p, F10p, and F20p, respectively, after the standard notation of Clive



ASSAY FORMAT:
DOSING:
- L5178Y cells were counted with a Coulter Counter model ZM and resuspended in Fisher's Medium with 5 % horse serum (F5p) at a concentration of 10^6 cells/ml
- After one hour, 6 ml of the cell suspension were pipetted into each culture tube
- Cells were then centrifuged down and the supernatant medium drawn off
- Cells were then resuspended in F0p containing the desired concentration of nitroguanidine
- For groups treated without metabolic activation, 9.7 ml were added, while those treated without metabolic activation received 5.7 ml
- 300 µl of horse serum were added to bring the serum concentration to 3%
- Positive controls were added
- Negative controls were prepared for both, the metabolic activation series and the nonactivated series
- The groups of the metabolic activation series received 3 ml of cofactor mixture and 1 ml of freshly thawed S-9 suspension
- The cofactor mixture also contained nitroguanidine in some cases to achieve the desired final compound concentration
- The low serum concentration (3%) was intended to reduce the possible interaction (and inactivation) of test compounds with the serum proteins
- These cultures were maintained at 37 °C on a roller drum for 4 h, washed twice with Fisher's Medium containing 10% horse serum (F10p), resuspended in 20 ml of F10p, and returned to the roller drum
- 10% serum in medium provided for rapid growth in suspension culture

CULTURING:
Nonselective:
- Soft agar cloning was used to determine the percentage of viable cells (viable count) and thymidine kinase negative mutants (mutant count) in each control and treated culture
- To determine the percentage of viable cells, a potion of each freshly diluted culture (3x10^5 cells/ml) was further diluted to 600 cells/ml in F20p
- 1 ml of this suspension was diluted in 105 ml of F20p containing 0.4% agar at 37 °C
- After vigorous mixing, this suspension of 5.7 cells/ml was dispensed into three 100 mm petri dishes (33 ml/dish)
- The extra 5 ml were provided to compensate for medium that foamed or adhered to the sides and thus could not readily be dispensed into petri plates
- The agar was allowed to harden at room temperature in the laminar flow hood (about 10 minutes)

Selective:
- To determine the percentage of thymidine kinase negative mutants, a similar but selective cloning procedure was performed
- 10 ml of the 3x10^5 cells/ml suspension were diluted with 95 ml of F20p with 0.4% agar (final concentration) which contained 1 µg/ml of trifluorothymidine (TFT)
- TFT was used to arrest the growth of all cells that contained thymidine kinase
- After mixing, 33 ml of this 2.86x10^4 cells/ml suspension were placed into each of three 100 mm petri dishes
- After hardening, both the mutant and viable count dishes were incubated for 11 days at 37 °C in a humidified atmosphere of 95% air and 5% CO2
- The number of colonies on each plate was then determined using a Biotran II Automated Colony Counter with the size setting on zero



NUMBER OF ASSAYS:
- Initial and confirmatory

Evaluation criteria:

Validity criteria: see below -> "Any other information on materials and methods incl. tables"

Criteria for positive or negative response:
- An individual treatment is considered positive if the assay is valid, the cell survival is at least 10% of controls, and the induced mutant frequency is at least three times (p < 0.01) the standard error of that mutant frequency
- A test compound is considered mutagenic if it yields a correlated positive dose response through several (usually three) treatment concentrations
- A compound is considered nonmutagenic in this system if a valid assay does not yield a positive response and the limits of compound solubility (up to 5 mg/ml) or 90% reduction in cell survival has been reached
- Normally, a determination of mutagenic potential is not made on the basis of only one assay; both positive and negative assays are confirmed

Statistics:

- Variance and standard error (SE) of the mutant frequency are calculated by using the mean selective and nonselective plate counts and with an assumed dilution variance of 10%.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Even at the highest doses, cytotoxicity was mild, with relative cell survivals of 70 % or greater in all cases.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

INITIAL ASSAY:
- Concentration range: 4.0 - 0.1 mg/ml
- Absolute suspension growth and absolute cloning efficiency for the negative controls were within prescribed limits
- The apparent greater-than-100% absolute cloning efficiency was the result of the time delay between cell counting and dilution of these samples
- Both positive controls produced a statistically significant mutagenic response as required for a valid assay
- Nitroguanidine did not produce significant mutagenic activity at any concentration tested

CONFIRMATORY ASSAY:
- Concentration range: 4.0 - 1.0 mg/ml
- Valid in terms of cell growth, cloning efficiency, and positive control-induced mutagenic activity
- No statistically significant mutagenic activity was induced by nitroguanidine

Table 1: Cell Survival data from the Initial Assay

Treatment	S-9	Cell Count		ASG^a	RSG^b	VC^c	ACE^d	RCE^e	RCS^f
		Day 1	Day 2
		(x 10³/ml)
Control^g	-	1032	1347	15.30	100	250	132	100	100
EMS 0.32  mg/ml	-	1059	1320	15.40	101	217	115	87	88
NG   4.0    mg/ml	-	891	1254	12.60	82	276	146	110	90
NG   2.0    mg/ml	-	848	1484	13.72	90	228	120	91	81
NG   1.0    mg/ml	-	949	1337	14.40	94	245	129	98	92
NG   0.5    mg/ml	-	994	1333	14.52	95	246	130	98	92
NG   0.1    mg/ml	-	1029	1359	15.30	100	259	137	104	104
NG   0.05  mg/ml	-	1155	1271	16.38	107	270	143	108	116
NG   0.01  mg/ml	-	1112	1257	15.54	102	246	130	98	100
Control	+	880	1394	13.34	100	235	124	100	100
2AAF 0.32  mg/ml	+	77	136	0.45	3	28	15	12	0
NG   3.66  mg/ml	+	756	1360	11.25	84	257	136	109	92
NG   2.0    mg/ml	+	819	1299	11.61	87	268	142	114	100
NG   1.0    mg/ml	+	867	1221	11.89	88	275	146	117	104
NG   0.5    mg/ml	+	808	1394	12.42	93	250	132	106	99
NG   0.1    mg/ml	+	906	1326	13.20	98	249	132	106	104
NG   0.05  mg/ml	+	1002	1147	12.54	94	232	123	99	93
NG   0.01  mg/ml	+	929	1127	11.78	86	307	162	131	113

^aAbsolute suspension growth = Total fold increase in suspension culture

^bRelative suspension growth = treated/control x 100

^cViable clone Count = mean of 3 plated

^dAbsolute Cloning Efficiency = viable clone count/189 x 100

^eRelative Cloning Efficiency = treated/control x 100

^fRelative Cell Survival = absolute cell survival treated/absolute cell survival control x 100

^gMean values from both negative controls

 

Table 2: Mutagenesis Data from the Initial Assay

Treatment	S-9	RCS^a	MF^b	IMF^c	SE^d	IMF/SE^e
			(x 10^-6)	(x 10^-6)	(x 10^-6)
Control^g	-	100	23.9
EMS 0.32  mg/ml	-	88	50.1	26.2	6.66	3.93
NG   4.0    mg/ml	-	90	15.9	- 7.9
NG   2.0    mg/ml	-	81	14.3	- 9.5
NG   1.0    mg/ml	-	92	17.7	- 6.2
NG   0.5    mg/ml	-	92	14.1	- 9.8
NG   0.1    mg/ml	-	104	11.6	- 12.3
NG   0.05  mg/ml	-	116	20.5	- 3.4
NG   0.01  mg/ml	-	100	24.7	0.8	3.69	0.21
Control	+	100	46.0	0.0
2AAF 0.32  mg/ml	+	0	17.86	132.6	33.52	3.96
NG   3.66  mg/ml	+	92	34.0	- 12.0
NG   2.0    mg/ml	+	100	30.8	- 15.2
NG   1.0    mg/ml	+	104	30.0	- 15.9
NG   0.5    mg/ml	+	99	38.5	- 7.6
NG   0.1    mg/ml	+	104	24.9	- 21.1
NG   0.05  mg/ml	+	93	33.3	- 12.7
NG   0.01  mg/ml	+	113	41.1	- 5.0

^aRelative Cell Survival = absolute cell survival treatment/absolute cell survival controls x 100

^bMutant Frequency = mutant clone count/viable clone count x dilution factor

^cInduced Mutant Frequency = treated mutant frequency – control mutant frequency

^dStandard Error is calculated only when the IMF > 0

^eRatio of the IMF to SE > 3 indicates a positive treatment response

^fValues from both negative controls

Table 3: Cell Survival Data from the Confirmatory Assay

Treatment	S-9	Cell Count		ASG^a	RSG^b	VC^c	ACE^d	RCE^e	RCS^f
		Day 1	Day 2
		(x 10³/ml)
Control^g	-	1178	1162	21.65	100	169	89	100	100
EMS 0.32  mg/ml	-	705	1610	12.96	60	160	84	95	57
NG   4.0    mg/ml	-	841	1576	14.84	69	177	94	105	72
NG   2.0    mg/ml	-	1028	1657	18.70	86	163	86	97	83
NG   1.0    mg/ml	-	1074	1649	19.80	91	172	91	102	93
Control	+	793	1590	14.05	100	173	92	100	100
2AAF 0.32  mg/ml	+	589	1492	10.00	71	169	89	98	70
NG   3.84  mg/ml	+	630	1552	10.92	78	157	83	91	71
NG   2.0    mg/ml	+	689	1602	12.19	87	130	69	75	65
NG   1.0    mg/ml	+	654	1632	11.88	85	157	83	91	77

^aAbsolute suspension growth = Total fold increase in suspension culture

^bRelative suspension growth = treated/control x 100

^cViable clone Count = mean of 3 plated

^dAbsolute Cloning Efficiency = viable clone count/189 x 100

^eRelative Cloning Efficiency = treated/control x 100

^fRelative Cell Survival = absolute cell survival treated/absolute cell survival control x 100

^gMean values from both negative controls

 

Table 4: Mutagenesis Data from the Confirmatory Assay

Treatment	S-9	RCS^a	MF^b	IMF^c	SE^d	IMF/SE^e
			(x 10^-6)	(x 10^-6)	(x 10^-6)
Control^g	-	100	60.8
EMS 0.32  mg/ml	-	57	395.8	334.6	45.33	7.38
NG   4.0    mg/ml	-	72	42.1	- 18.7
NG   2.0    mg/ml	-	83	44.3	- 16.5
NG   1.0    mg/ml	-	93	67.3	6.5	8.95	0.73
Control	+	100	59.4
2AAF 0.32  mg/ml	+	70	11.82	58.8	14.63	4.02
NG   3.84  mg/ml	+	71	58.7	- 0.7
NG   2.0    mg/ml	+	65	63.1	3.7	9.07	0.41
NG   1.0    mg/ml	+	77	65.8	6.4	8.97	0.71

^aRelative Cell Survival = absolute cell survival treatment/absolute cell survival controls x 100

^bMutant Frequency = mutant clone count/viable clone count x dilution factor

^cInduced Mutant Frequency = treated mutant frequency – control mutant frequency

^dStandard Error is calculated only when the IMF > 0

^eRatio of the IMF to SE > 3 indicates a positive treatment response

^fValues from both negative controls

 

Conclusions:

The mutagenic potential of nitroguanidine was evaluated in the mouse lymphoma thymidine kinase forward mutation assay. Nitroguanidine was not mutagenic under the conditions of this study.

Executive summary:

The mutagenicity of nitroguanidine was evaluated in a mouse lymphoma (cell line: L5178Y) forward mutation study consisting of an initial and a confirmatory assay. The results of this study indicated that nitroguanidine was not mutagenic in the mouse lymphoma test system. Both assay met the acceptance criteria for cloning efficiency, cell replication, positive control responses, and maximum test compound concentrations.

Spontaneous mutation rates were well within published values.

The highest concentration tested was 4 mg/ml which is nearly the limit of solubility for nitroguanidine.

Metabolic activation with rat liver S-9 did not significantly alter either cytotoxicity or mutagenicity of nitroguanidine. Even at the highest doses, cytotoxicity was mild, with relative cell survivals of 70% or greater in all cases.

Based on the results of this study it can be concluded that Nitroguanidine was not mutagenic under the conditions of this study.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo study nitroguanidine was non-mutagenic in an in vivo Drosophila melanogenaster sex-linked recessive lethal assay according to EPA OPPTS 870.5275.

Link to relevant study records

Reference

Endpoint:

in vivo insect germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 1985 - December 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5275 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)

Deviations:

yes

Remarks:

1. CS males were dosed in conc. based on results of preliminary study. 2. Tot. number flies in test and in control group was less than 8000. 3. DMSO as a solvent was avoided as recomm. by the EPA. These deviations had no effect on the outcome of the study

GLP compliance:

yes

Type of assay:

Drosophila SLRL assay

Specific details on test material used for the study:

Test substance supplier Aunflower AAP

Species:

Drosophila melanogaster

Strain:

other: Canton-S (CS), wild-type stock and Basc, laboratory stock

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: insectary of Letterman Army Institute of Research; the original stock colonies were obtained from the Mid-American Drosophila stock center, Bowling Green State University, Bowling Green, Ohio
- Housing: all insect colonies were reared in polypropylene bottles; those selected for SLRL testing were housed in glass vials
- Diet (e.g. ad libitum): standard medium consisiting of cornmeal (NSCO Chemicals), unsulphured molasses (Ingredient Technology Corp.), Yeast (Nabisco Brands, Inc.), and nutrient agar (Moorhead & Co., Inc.)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2 °C
- Humidity (%): 57 +/- 8%
- Photoperiod (hrs dark/hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

- solution of 1% fructose in water was found to be an appropriate vehicle for the nitroguanidine

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The solubility of nitroguanidine in water is relatively high compared to that of most explosives. Preliminary studies were conducted to test potentila dosages for toxicity to flies and their ability to feed and digest nitroguanidine in an appropriate medium
- A feeding solution vehicle of 1.0% fructose in deionized water was selected for the study

Duration of treatment / exposure:

72 h

Frequency of treatment:

Dosing was continous for 72 h. Flies were transferred every 24 hours to vials containing fresh compound solutions

Post exposure period:

8 days

Remarks:

Doses / Concentrations:
2.08 µg/ml - 20.8 µg/ml
Basis:
nominal conc.

No. of animals per sex per dose:

25 surviving CS males (wild type) were selected after feeding on the dosing solution and were scored by mating with Basc virgin females (ratio males:females = 1:3). At days 1,4, 6, and 8 after dosing the CS male was transferred to successive groups of 3 Basc virgin females. 4 replicates
Total number of test flies examined in the test group was 6913, for the negative control group, the number of flies was 7328.

Control animals:

yes, concurrent vehicle

Positive control(s):

- 1.0 mM ethylmethane sulfonate solution formulated with 1.0% fructose in water

Tissues and cell types examined:

x-chromosomes

Details of tissue and slide preparation:

no details reported

Evaluation criteria:

- Scoring of the mutants resulting from positive control exposure was based on mating 5 CS males in the same manner as males treated with the test compound (4 replicates).
- After sufficient numbers of flies had emerged, a maximum of 25 (minimum of 5) kidney-shaped red-eyed F1 females were selected at random and mated with their sibling white-body, bar-shaped, apricot-eyed males.
- After 2 to 3 weeks the F2 progeny were examined and scored for the absence of round, red-eyed males, which would indicate that a lethal mutation had taken place in the treated male.
- Confirmation of a lethal mutation was obtained by conducting an F3 cross from each vial scored as a lethal mutation

Statistics:

The testing was designed to examine approx. 2500 X-chromosomes in each of 4 replications, thereby yielding a total of 8000 to 10000 X-chromosomes for examination. Vials without F2 progeny or fewer than 5 progeny (F2) were scored as failures. The BMDP (Biomedical Programs) computer package was used to perform the analyses. Based on the number of lethal and nonlethal offspring for each male, by combining all replicates, the mutation frequency of nitroguanidine was compared to that of the control by Fisher's exact test for each of the four broods seperately and for the combined broods. All statistical tests were conducted at the 0.05 level of significance.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
The frequencies of spontaneous mutation for nitroguanidine and the negative control were 0.188% and 0.096% based on 6913 and 7318 X-chromosomes, respectively. The mutation frequencies for the positive control, 1 mM ethylmethane sulfonate, was 17.8%. The mutation frequencies for each compound are presented in table 1. The mutation frequencies for each brood for nitroguanidine and the negative control are presented in table 2. No significant difference was detected between the mutation frequency of the negative control and the nitroguanidine with the Fisher's exact test (p = 0.1799). Also, no significant differences were detected between the negative control and the nitroguanidine for the data of broods 1, 2, 3, and 4 (table 2).

Table 1: Percent mutation frequencies in the Sex-linked Recessive Lethal Assay of nitroguanidine*

Compound	1	2	3	4	Total Mutations	Percent Mutations
Nitroguanidine	0/1115	5/1231	4/2352	4/2215	13/6913	0.188
Negative Control	1/1481	1/1529	3/2155	2/2153	7/7318	0.096
Positive Control	36/166	39/284	56/395	70/284	201/1129	17.80

* Data are recorded as number of SLRL events/numbers of X-chromosomes tested

 

Nitroguanidine:            25 maleDrosophila melanogenasterflies(CS strain) formed the P generation

 

Negative Control:          25 maleDrosophila melanogenasterflies(CS strain) formed the P generation

 

Positive Control:            5 maleDrosophila melanogenasterflies(CS strain) formed the P generation

 

 

Table 2: Fisher’s Exact Test for Significance of the difference between nitroguanidine and negative control in Sex-Linked Recessive Lethal Assay

	Brood number
Compound	1	2	3	4
Nitroguanidine	3/2055	3/1815	5/1727	2/1316
Negative Control	2/2094	2/1901	1/1844	2/1479
Positive Control	44/336	88/291	63/316	6/186
p values	0.6845	0.6805	0.1138	1.0000

Nitroguanidine:            Nitroguanidine was dissolved in a 1% fructose solution in deionized water. Date are from 25 male Drosophila melanogenaster flies (CS strain) x 4 replicates mated with 3 Basc strain female flies each.

 

Negative Control:         1% fructose solution in deionized water. Date are from 25 Drosophila melanogenaster flies (CS strain) x 4 replicates mated with 3 Basc strain female flies each.

 

Positive Control:          1 mM ethylmethane sulfonate and 1% fructose in deionized water. Date are from 5 Drosophila melanogenaster flies (CS strain) x 4 replicates mated with 3 Basc strain female flies each.

 

Conclusions:

Nitroguanidine was non-mutagenic following 72-hour feeding exposure to concentrations of nitroguanidine ranging from 2.08 µg/ml to 20.8 µg/ml.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of nitroguanidine in the Drosophila melanogenaster Sex-Linked Recessive Lethal Assay.

Nitroguanidine has been reported to produce significant damage in Chinese hamster fibroblasts (see IUCLID 7.6.1, "556-88-7_Nitroguanidin_chromosome_tests_hamster_cells_1976.pdf").

However, studies from this laboratory have indicated, that nitroguanidine is not mutagenic in the mouse lymphoma forward mutation assay (see IUCLID 7.6.1, "556-88-7_Nitroguanidin_mutagenic_potential_mouse_lymphoma_1987.pdf"), the Chinese hamster ovary sister chromatid exchange assay (see IUCLID 7.6.1, "556-88-7_Nitroguanidin_genetic_toxicology_1988.pdf"), or in the Ames Salmonella / mammalian microsome assay (see IUCLID 7.6.1, "556-88-7_Nitroguanidin_genetic_toxicology_ames_test_1988.pdf").

The results of this study confirm in an in vivo model for genetic toxicity the previous findings that nitroguanidine has no mutagenic potential at doses which approach the limits of solubility in the appropriate test systems.

The results of this study indicate that nitroguanidine is not mutagenic when evaluated in the Drosophila melanogenaster Sex-Linked Recessive Lethal Assay.following 72-hour feeding exposure to concentrations of nitroguanidine ranging from 2.08 µg/ml to 20.8 µg/ml.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Nitroguanidine was tested non-mutagenic in an in vitro gene mutation assay in bacteria (Ames test), an in vitro cytogenicity study in mammalian cells (SCE), an in vitro gene mutation assay in mammalian cells (mouse lymphoma assay), an in vitro chromosome aberration assay (human lymphocytes), and an in vivo Drosophila melanogenaster sex-linked recessive lethal assay. All these studies were reliable, relevant and adequate. The in vitro assays were conducted with concentrations close to the maximum solubility of nitroguanidine.

In addition there were findings in a less reliable publication indicating lack of mutagenicity in a DNA repair assay in E. coli, in a reverse mutation assay in S. typhimurium, and in a mitotic recombinogenic test with yeast. The tests were carried out with and without metabolic activation.

A pre OECD/GLP in vitro chromosomal aberration assay was found to contradict these results (Ishidate and Odshima, 1977). This CA assay was designed as a rapid in vitro screening test to assess 134 chemicals in a Chinese hamster fibroblast cell line (CHL) without metabolic activation. Nitroguanidine was tested positive at a concentration of 4 mg/ml (close to the limit of solubility) following 24 h exposure. 26% of the metaphases were found to have chromosomal aberrations. The documentation of this publication is not sufficient for assessment. No guideline was followed. The method was reported in low detail. Only 100 metaphases were counted (OECD 473 recommends counting at least 200). No details on the test compound were provided. No dose dependency was reported. An extended exposure time of 24 h was used. In comparison, Harbell et al. (1988) found that sister chromatid exchange (SCE) could not be evaluated after an extended exposure time of 21.5 h (CHO cells, 4 mg/ml nitroguanidine). In the SCE-assay nitroguanidine proved to be very toxic at that dose when exposed for 21.5 h. 91% of the metaphases detected were from first-division cells, and even those that showed some differential staining had not gone through two complete replication cycles in the BrdU-containing medium. Instead, these cells appeared to have been in the S phase when nitroguanidine and BrdU were added and went on to complete this cycle. After the second S phase, these cells contained a mixture of thymidine, thymidine and BrdU, and BrdU containing strands so that they were not scalable for SCEs. Examination of the metaphases from this dose group did not reveal the chromosomal aberrations reported by Ishidate and Odshima (1977). Using 2 mg nitroguanidine/ml for 21.5 h also induced some toxicity but no SCE. Below 2 mg/ml neither toxicity nor SCE was reported. The same effect was reported in a mouse lymphoma test conducted by Harbell et al. (1987). Nitroguanidine was found to be very cytotoxic when mouse lymphoma L5178Y cells were exposed for 24 h. Hence, the chromosome aberration reported by Ishidate and Odshima was most likely caused by an unsuitable exposure regime and is no evidence for a mutagenic property of nitroguanidine. This assumption has been confirmed by a second chromosome aberration test in human lymphocytes according to OECD 473 (Hofman-Huther, H. (2014)), which reported a lack of genotoxicity up to the maximum solubility.

Justification for selection of genetic toxicity endpoint
As the overall conclusion from the test battery of relevant and valid genetic toxicity studies, nitroguanidine is not genotoxic.

Justification for classification or non-classification

Nitroguanidine was found to be non-mutagenic.