Propargite

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th May 1994 to 16th September 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

other: micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Propargite

EC Number:

219-006-1

EC Name:

Propargite

Cas Number:

2312-35-8

Molecular formula:

C19H26O4S

IUPAC Name:

propargite

Test material form:

liquid

Details on test material:

- Storage condition of test material: room temperature, protected from exposure to light

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Frederick, MD, USA
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 24.9-34.8 g (males); 22.8-30.3 g (females)
- Assigned to test groups randomly: yes
- Housing: up to five per cage in plastic autoclavable cages
- Diet: certified laboratory rodent chow ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 74 ± 6 ºF
- Humidity: 50 ± 20 %
- Photoperiod: 12 hours light/12 hours dark

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

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

Duration of treatment / exposure:

One IP injection at a constant volume of 10 mL/kg bw

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

37.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

Examinations

Tissues and cell types examined:

Femur bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice time, five animals per sex per treatment were sacrificed and, immediately after sacrifice, the femurs exposed and the bone marrow aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred into a centrifuge tube containing 1 mL fetal bovine serum and pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant drawn off. The cells were resuspended by aspiration and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol, stained with May-Gruenwald-Giesma and permanently mounted.

Evaluation criteria:

1000 polychromatic erythrocytes were scored for the presence of micronuclei (defined as round, darkly staining nuclear fragments having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte). The number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes counted was also recorded per 100 erythrocytes.

The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each animal and treatment group. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.

The test material was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time. The test material was considered to be positive if a statistically significant treatment-related increase in micronucleated polychromatic erythrocytes was observed relate to the vehicle control. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, this assay was considered a suspect or unconfirmed positive.

Statistics:

Statistical significance was determined using the Kastenbaum-Bowman table which are based on binomial distribution. All analyses were performed separately for each sex.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): not statistically increased regardless of dose level or bone marrow collection time relative to respective vehicle controls (see Table 2).
- Ratio of PCE/NCE (for Micronucleus assay): 18-51 % reduction in male and female mice 48 and 72 hours following treatment with 75 and 150 mg/kg and in female mice 24 hours following treatment with 150 mg/kg relative to their respective vehicle controls (see Table 2).

Any other information on results incl. tables

Pilot study

Mortality occurred within one to two days of dose administration as follows: all animals at 2500 mg/kg, 2/2 males at 1600 and 1000 mg/kg and 1/2 males at 500 mg/kg. Clinical signs included lethargy in mice treated with 100, 500, 1000 and 2500 mg/kg and crusty eyes in one 100 mg/kg male.

Toxicity study

Mortality occurred within two days of dose administration as follows: 4/5 males and 5/5 females at 800 mg/kg, 2/5 males and 3/5 females at 500 mg/kg, 3/5 males and 4/5 females at 300 mg/kg and 2/5 males and 1/5 females at 200 mg/kg. Clinical signs, which were noted on the day following dose administration included lethargy in both males and females at all dose levels, diarrhea in one female at 300 mg/kg and crusty eyes in one male at 500 mg/kg. The LD_50/3 was calculated to be approximately 290 mg/kg. The high dose for the micronucleus test was set at 150 mg/kg which was approximately 50% of the LD_50/3.

Table 1: Body weights and mortality from toxicity study

		Group mean body weights (g)			% change
Treatment	Sex	Pre-treatment	Day 1	Day 3	Day 1	Day 3	Mortality
200 mg/kg	M	27.3 ± 1.3	28.6 ± 1.8	25.7 ± 2.8	4.8	-5.9	2/5
	F	27.7 ± 0.6	26.1 ± 1.1	24.2 ± 0.7	-5.8	-12.6	1/5
300 mg/kg	M	30.8 ± 2.8	29.2 ± 2.1	26.7 ± 2.3	-5.2	-13.3	3/5
	F	27.8 ± 0.4	26.8 ± 1.0	28.9*	-3.6	-4.0	4/5
500 mg/kg	M	30.8 ± 1.8	30.4 ± 1.8	29.9 ± 2.9	-1.3	-2.9	2/5
	F	27.7 ± 1.3	27.5 ± 0.8	26.9 ± 1.8	-0.7	-2.9	3/5
800 mg/kg	M	30.7 ± 1.2	30.0 ± 2.0	32.5*	-2.3	5.9	4/5
	F	28.0 ± 1.3	27.4 ± 1.1	**	-2.1	**	5/5
2500 mg/kg	M	25.7 ± 0.9	22.3*	**	-13.2	**	5/5
	F	23.7 ± 0.6	**	**	**	**	5/5

*standard deviation not available due to single surviving animal

**no data due to mortality

Table 2: Summary of results from micronucleus assay

					Micronculeated polychromatic erythrocytes
Treatment	Sex	Time (hr)	Number of mice	PCE/total erythrocytes	Number per 1000 PCEs (mean ± S.D.)	Number per PCE scored
Vehicle	M	24	5	0.48	0.4 ± 0.55	2/5000
		48	5	0.50	0.2 ± 0.45	1/5000
		72	5	0.54	0.2 ± 0.45	1/5000
	F	24	5	0.61	0.0 ± 0.00	0/5000
		48	5	0.50	0.4 ± 0.55	2/5000
		72	5	0.63	0.0 ± 0.00	0/5000
37.5 mg/kg	M	24	5	0.49	0.2 ± 0.45	1/5000
		48	5	0.45	0.0 ± 0.00	0/5000
		72	5	0.59	0.2 ± 0.45	1/5000
	F	24	5	0.56	0.6 ± 0.55	3/5000
		48	5	0.51	0.2 ± 0.45	1/5000
		72	5	0.67	0.2 ± 0.45	1/5000
75 mg/kg	M	24	5	0.56	0.8 ± 1.30	4/5000
		48	5	0.33	0.4 ± 0.55	2/5000
		72	5	0.28	0.2 ± 0.45	1/5000
	F	24	5	0.56	0.2 ± 0.45	1/5000
		48	5	0.40	0.2 ± 0.45	1/5000
		72	5	0.46	0.0 ± 0.00	0/5000
150 mg/kg	M	24	5	0.52	0.2 ± 0.45	1/5000
		48	5	0.40	0.0 ± 0.00	0/5000
		72	5	0.38	0.2 ± 0.45	1/5000
	F	24	5	0.50	0.6 ± 0.89	3/5000
		48	5	0.37	0.2 ± 0.45	1/5000
		72	5	0.31	0.0 ± 0.00	0/5000
Positive control	M	24	5	0.41	8.2 ± 6.61	41/5000
	F	24	5	0.53	9.8 ± 4.32	49/5000

Historical control data

Table 3: Negative control animals

	Ratio of PCE/total erythrocytes		MPCE/1000 PCE scored
Parameter	Males	Females	Males	Females
Mean	0.57	0.60	0.41	0.47
Standard Deviation	0.09	0.08	0.76	0.75
Range	0.12 - 0.85	0.09 - 0.86	0 to 8	0 to 5

Table 4: Positive control animals

	Ratio of PCE/total erythrocytes		MPCE/1000 PCE scored
Parameter	Males	Females	Males	Females
Mean	0.56	0.59	12.01	11.59
Standard Deviation	0.10	0.11	6.53	5.41
Range	0.12 - 0.85	0.04 - 0.80	1 to 51	2 to 40

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Under the conditions of the test, the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.

Executive summary:

Male and female ICR mice were exposed to 37.5, 75 or 150 mg/kg bw of the test material which was administered in a total volume of 10 mL/kg as a single IP injection. The high does level was calculated to be approximately 50 % of the LD_50(3day). Corn oil was used as the vehicle. Mortality was observed in 2/20 males and 1/20 females receiving 150 mg/kg. Clinical signs following dose administration included lethargy and diarrhea in mice treated with 75 and 150 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronculeated polychromatic erythrocytes. Relative to their respective vehicle controls, an 18 to 51 % reduction in the frequency of polychromatic erythrocytes was observed in bone marrow from male and female mice following treatment with 75 and 150 mg/kg. This reduction demonstrates that the test material was transported to the bone marrow target tissue resulting in an inhibition of erythropoiesis. No significant increases in microncuelated polychromatic erythrocytes were observed in male or female ICR mice at 24, 48 or 72 hours after dose administration relative to their respective vehicle controls. The results of the assay indicate that under the conditions of the test, the test material did not induce a significant increase in micronucelated polychromatic erythrocytes in either male or female ICR mice. The test material was determined to be negative in the mouse micronculeus assay.