Nitrapyrin

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

Study period:

03 December 1984 to 11 March 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A GLP study conducted to sound scientific principles with a sufficient level of detail to assess the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Nitrapyrin

EC Number:

217-682-2

EC Name:

Nitrapyrin

Cas Number:

1929-82-4

Molecular formula:

C6H3Cl4N

IUPAC Name:

2-chloro-6-(trichloromethyl)pyridine

Test material form:

solid: crystalline

Details on test material:

- Appearance: white crystalline solid
- Storage conditions of test material: in the dark at room temperature

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 24 - 31 g (males); 22 - 30 g (females)
- Housing: housed in groups of 5 animals of the same sex in polypropylene cages with solid floors and wire mesh lids
- Assigned to test groups randomly: yes (randomised to groups of 5 with approximately equal group weights)
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: short period (not specified)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23 °C
- Humidity (%): 50 ± 5 %
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours of darkness / 12 hours of light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test material was dissolved in corn oil by stirring at room temperature.
For the initial range-finder solutions of 20, 30 and 40 mg/mL were prepared; for the detailed range-finder test solutions of 16, 24, 32 and 40 mg/mL were prepared, and for the main micronucleus assay a solution of 32 mg/mL was prepared. In all cases the dose volume was 25 mL/kg

Duration of treatment / exposure:

Animals were treated by oral gavage

Frequency of treatment:

Single administration

Post exposure period:

Groups were killed 24, 48 and 72 hours following treatment.

Doses / concentrations

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 males and 15 females per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 80 mg/kg (3.2 mg/mL and a dose volume of 25 mL/kg)

Examinations

Tissues and cell types examined:

One femur from each animal was exposed, removed, cleaned of adherent muscle and connective tissue so that the shank could be removed from the ends by using fine clippers or scissors. Disposable centrifuge tubes containing 1 mL of foetal bovine serum (FBS) were labelled with the animal numbers.
Approximately 0.5 mL of FBS was drawn into a 1 mL syringe with a fresh needle, and this was used to aspirate the contents of the femur into the centrifuge tube.

Details of tissue and slide preparation:

The disposable centrifuge tubes containing the contents of the femurs were centrifuged at 2000 rpm for 2 - 3 minutes, the serum was carefully decanted to leave one or two drops of serum and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of 4 labelled slides. The end of a clean slide was placed across the drop, and a smear made by drawing the clean slide along the labelled slide.
Slides were allowed to air-dry and were stored for at least 48 hours before staining. In brief, two slides from each set of four were taken (the others were kept in reserve but, on this occasion were not needed) and fixed for 5 minutes in absolute ethanol. After rinsing several times in water, the slides were then stained for 10 minutes in Gurr's Giemsa R66 stain diluted 1:6 (v/v) in distilled water. Slides were rinsed, and allowed to dry thoroughly before clearing in xylene for 3 minutes. When dry, slides were finally mounted with coverslips.

Statistics:

The numbers of micronucleated PCE and NCE of groups treated with test material or positive control were compared to vehicle controls using the chi-squared method.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mice treated with test material exhibited PCE/NCE ratios which were similar to vehicle controls at 24 hours. There was some indication of toxicity as the ratios tended to be lower than controls at 48 and, in particular, at 72 hours where the control PCE/NCE range was 0.42 - 0.82, but six test material-treated mice had ratios in the range 0.09 - 0.51. The numbers of micronucleated PCE and NCE were generally similar to those seen in controls. Group mean frequencies exceeded controls for micronucleated PCE at 72 hours and micronucleated NCE at 48 hours, but the differences were small and not significant by chi-squared analysis. In all other cases the mean frequencies of micronucleated PCE and NCE were lower than in controls, and for micronucleated NCE at 24 and 72 hours these decreases were significant by chi-squared analysis.
All vehicle control mice exhibited a normal PCE/NCE ratio in the region of 1.0 (0.4 - 2.4 was the range).
Mice treated with the positive control chemical exhibited much lower PCE/NCE ratios (0.12 - 0.38) indicating some bone marrow toxicity. In 7 out of the 10 CPA-treated mice, numbers of micronucleated PCE clearly exceeded those seen in vehicle controls, such that the mean frequency (27.3/1000) was 5x the highest mean frequency seen in controls. Chi-squared analysis on the total counts revealed the increased numbers of micronuclei to be significant. In addition, in at least 8 of the 10 CPA-treated mice, numbers of micronucleated NCE also clearly exceeded those seen in vehicle controls, such that total numbers of micronucleated NCE in the CPA group were significantly different from controls by chi-squared analysis.

Any other information on results incl. tables

Table 1: Summary of group mean data for test material, vehicle and positive control

Treatment group	Kill time (hours)	Sex	Mean ratio (PCE/NCE)	Mean frequency of micronucleated PCE (/1000)		Mean frequency of micronucleated NCE (/1000)
				per sex	per group	per sex	per group
Vehicle control	24	M	1.30	5.20	5.69	6.17	7.70
		F	1.54	6.19		9.24
	48	M	0.85	4.80	4.50	4.01	3.79
		F	0.71	4.20		3.57
	72	M	0.64	1.80	2.30	2.62	2.73
		F	0.64	2.80		2.84
Test material	24	M	1.47	5.20	4.50	6.36	5.50
		F	0.94	3.80		4.65
	48	M	0.64	3.60	4.00	5.20	4.61
		F	0.68	4.40		4.03
	72	M	0.43	2.00	2.40	2.18	1.81
		F	0.55	2.80		1.43
Positive control	48	M	0.17	13.2	27.30	9.61	13.09
		F	0.28	41.4		16.56

Applicant's summary and conclusion

Conclusions:

Interpretation of results: Negative
Under the conditions of the study, the test material was not able to induce micronuclei in the polychromatic or normochromatic erythrocytes of the bone marrow of mice treated with the maximum tolerated dose of 800 mg/kg.

Executive summary:

The clastogenic potential of the test material was investigated in a study which was conducted under GLP conditions and following a method similar to that which is outlined in the standardised guideline OECD 474.

During the study, the test material was evaluated for its ability to induce micronuclei in mice. A maximum tolerated dose of 800 mg/kg was established and this was administered to three groups of mice (5 male and 5 females per group). Test material was administered in corn oil by oral gavage; corn oil alone was administered to control mice.

The three groups of mice were killed after 24, 48 and 72 hours, and slides prepared from bone marrow.

Some evidence of bone marrow toxicity was obtained, evidenced by a slight decrease in the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE). The test material did not produce an increase in micronuclei in either PCE or NCE. It is therefore concluded that the test material is not clastogenic in the micronucleus test.