1-nitroguanidine

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

1-nitroguanidine

EC Number:

209-143-5

EC Name:

1-nitroguanidine

Cas Number:

556-88-7

Molecular formula:

CH4N4O2

IUPAC Name:

N-nitroguanidine

impurity 1

Reference substance name:

Sodium sulphate

EC Number:

231-820-9

EC Name:

Sodium sulphate

Cas Number:

7757-82-6

Molecular formula:

Na2O4S

IUPAC Name:

disodium sulfate

impurity 2

Reference substance name:

Sodium nitrate

EC Number:

231-554-3

EC Name:

Sodium nitrate

Cas Number:

7631-99-4

Molecular formula:

HNO3.Na

IUPAC Name:

sodium nitrate

impurity 3

Reference substance name:

4,6-diamino-1,3,5-triazin-2(1H)-one

EC Number:

211-455-1

EC Name:

4,6-diamino-1,3,5-triazin-2(1H)-one

Cas Number:

645-92-1

Molecular formula:

C3H5N5O

IUPAC Name:

4,6-diamino-1,3,5-triazin-2(1H)-one

impurity 4

Reference substance name:

6-amino-1,3,5-triazine-2,4(1H,3H)-dione

EC Number:

211-456-7

EC Name:

6-amino-1,3,5-triazine-2,4(1H,3H)-dione

Cas Number:

645-93-2

Molecular formula:

C3H4N4O2

IUPAC Name:

6-amino-1,3,5-triazine-2,4(1H,3H)-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test substance supplier Aunflower AAP

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Test results

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).

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion

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.