Dimethyl methylphosphonate

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: scientifically acceptable publication

Data source

Reference

Reference Type:

publication

Title:

Reprotjuctive Toxicity of Dimethyl Methyl Phosphonate (DMMP) in the Male Fischer 344 Rat

Author:

Dunnick JK, Gupta BN, Harris MW and Lamb IV JC

Year:

1984

Bibliographic source:

Toxicology and Applied Pharmacology 72: 379-387

Materials and methods

Principles of method if other than guideline:

determination of the reproductive toxicity in male rats of DMMP after subchronic dosing (dominant lethal test)

GLP compliance:

no

Remarks:

well documented study

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl methylphosphonate

EC Number:

212-052-3

EC Name:

Dimethyl methylphosphonate

Cas Number:

756-79-6

Molecular formula:

C3H9O3P

IUPAC Name:

dimethyl methylphosphonate

Details on test material:

- Name of test material (as cited in study report): dimethyl methyl phosphonate (DMMP)
- Analytical purity: >99%
- Lot/batch No.: not specified; test substance was obtained from Stauffer Chemical Company, Weslport, Connecticut
- Other: the compound was analyzed by gas chromatography, thin-layer chromatography and by infrared, ultraviolet and nuclear magnetic resonance spectroscopy

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: obtained from Charles River Breeding Laboratories, Portage, Michigan,
- Age at study initiation: (P) 42 days of age on the first day of dosing (referred to as day 1)
- Weight at study initiation: see Table 1
- Housing: animals were housed individually
- Diet (e.g. ad libitum): NIH-31 laboratory feed
- Water (e.g. ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1±1.1
- Humidity (%): 50±5
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and dark cycle was provided

No additional data provided

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Each rat received 3.5 ml of dose solution/kg body wt for each day of dosing.
No additional data

Details on mating procedure:

- M/F ratio per cage: 1:2; on day 84, two untreated virgin female Fischer 344 rats were housed with each male rat.
- Length of cohabitation: 5 days; the dosing of male rats continued during the 5-day cohabitation period (days 84-88)
- Proof of pregnancy: sperm in vaginal smear] referred to as day 1 of pregnancy
The female rats cohabitated with the male rats for 5 days (Days 84-88) and, during this period, daily vaginal smears were made to detect the presence o( sperm. Any female that was sperm positive was separated from the male.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aqueous solutions of DMMP used for dosing the animals were prepared fresh each week and were found to be stable and within ±6% of the theoretical concentrations. (All doses of DMMP used in this study were soluble in water.)

Duration of treatment / exposure:

90 days (during a period of 90 days the animals received a total of 63 doses)

Frequency of treatment:

5 days per week

Details on study schedule:

- Age at mating of the mated animals in the study: 126 days for males and 142 days for females

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 250, 500, 1000 or 2000 mg/kg/day (only male animals were treated)
Basis:
actual ingested

No. of animals per sex per dose:

20 males and 40 females (only male animals were treated)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses were set based on the results of a previous 13-week subchronic toxicity study in Fischer 344 rats in which DMMP administered in com oil was found to be fatal at 4000 mg/kg; at 2000 mg/kg some of the animals survived, and at 1000 mg/kg all animals survived the dosing schedule (NTP, 1981)

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: no data

DETAILED CLINICAL OBSERVATIONS: no data

BODY WEIGHT: no data

OTHER:
- LH AND FSH HORMONE LEVELS: LH and FSH levels were determined by the radioimmunoassay kits and procedures provided by the National Hormone and Pituitary Program of the National Institute of Arthritis Metabolism Digestive Diseases

Oestrous cyclicity (parental animals):

not applicable

Sperm parameters (parental animals):

Parameters examined in all P male parental generations: after 90 days, the rats were killed over a 4-day period (days 91-94) by exposure to CO2, and histopathologic studies were performed to measure toxicity to the reproductive system and the genitourinary system; 25 male rats, randomly selected, were killed each day.

- Evaluation of sperm count, motility, and morphology: sperm samples from the caudal epididymis were used for evaluation of sperm count, sperm motility, and sperm morphology. The right epididymis was separated from the testis and weighed; a small sample of sperm from the caudal epididymis was teased into egg yolk medium at 37°C. A sample of this sperm preparation was placed on a slide, and the motility was calculated by counting all sperm in 20 fields (magnification of 40x) and categorizing them as either motile or non-motile (any movement versus no movement). Sperm motility was determined within 5 min after the animal was killed.

A 20- to 35-mg piece of the right caudal epididymis was minced and evenly distributed with a Pasieur pipel in 2 ml of phosphate-buffered saline. Approximately 1 ml of this sperm suspension was stained with 1% eosin Y for 45 min, and then one drop was spread on a microscope slide, air-dried, and cover-slipped. These slides were used to evaluate sperm morphology at 400x. Sperm were then classified as normal or abnormal. Finally, 0.5 ml of the remaining sperm solution was diluted in 2 ml phosphate-buffered saline and heated in boiling water for 30 sec to kill the sperm. Sperm counts were made from this preparation with a hemocytometer.

Litter observations:

Not applicable

Postmortem examinations (parental animals):

MALE ANIMALS: at necropsy, blood was collected by cardiac punction in heparinized tubes and centrifuged; plasma was stored at -20°C for hormone determinations. Weights of kidneys, ventral prostate gland, testes, and epididymis were recorded and organ to body weight ratios calculated.

Tissue samples from kidneys, ventral prostate gland, testes, epididymis, coagulative gland, seminal vesicle, urinary bladder, pelvic, and extrapelvic urethra along with penile tissue, ductus deferens, preputial gland, urethral gland, bulbourethral gland, salivary gland, zymbal gland, thymus, pituitary gland, and tympanic bullae were fixed in buffered, pH 7.0, 10% formalin for histopathologic examination. These specimens were embedded in paraffin, sectioned 6 nm thick, and stained with hematoxylin and eosin. Part of the formalin-fixed testes was re-fixed in Bouin's solution and routinely processed for histopathologic examination.

FEMALE ANIMALS
For uteri analysis, all 200 female rats were killed by CO2 asphyxiation 14 days (Day 100) after the middle of the mating period (day 86)

Postmortem examinations (offspring):

Not applicable (see above)

Statistics:

For the dichotomous response (sperm-positive and pregnancy data), the Cochran-Armitage test was used to assess the significance of dose-response trends, and Fisher's exact test was used to make pairwise comparisons. For the remaining variables, dose-response trends were analyzed by Jonckheere's test and pairwise comparisons made by Mann-Whitney U tests. One-sided tests were employed throughout. A result was considered statistically significant if the P value was less than 0.05.

Reproductive indices:

see below

Offspring viability indices:

The uteri were removed and examined for number of live pups, number of dead pups, and number of resorptions.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All male rats dosed with 0, 250, 500, 1000, or 2000 mg/kg/day of test substance, 5 days per week for 90 days, survived the drug treatment, and no dose-related clinical signs or neurotoxicity was seen in any group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At the end of the 90-day dosing period, only the high-dose group (2000 mg/kg) showed a decrease in total body weight gain relative to the control group (Table 1).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not analyzed

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- Sperm analysis: the test substance was toxic to the reproductive system of the male rat (Table 2). Sperm motility and sperm count were reduced with increasing doses of test substance, and these changes were particularly significant in the high-dose group (p < 0.01). Morphologic abnormalities were seen in sperm from the high-dose rats (2000 mg/kg). These abnormalities were characterized by headless sperm and sperm heads without a hook or with a blunt hook.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating trial: the high-dose male rats (2000 mg/kg) failed to impregnate any female rats, even though there was evidence of copulation for 55% of the males (Table 3). The male rats in the 500 and 1000 mg/kg groups were able to impregnate some female rats and the 250 mg/kg group had a fertility index comparable to the control level. The number of live pups decreased with increasing dose of test substance. The mean number (±SEM) of resorptions per pregnant female for the 0, 250, 500, and 1000 mg/kg groups was 0.5 ± 0.6, 1.3 ± 1.0, 3.5 ± 1.0, and 3.1 ± 1.4, respectively. The percentage of resorptions (total number of resorptions/ total number of fetuses and resorptions) increased with increasing doses of test substance. Dead fetuses were seen only in two litters from the 500 mg/kg group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The test substance did not alter the organ to body weight ratio of the testes or prostate. There was a small increase in the kidney/body weight ratio and a decrease in the epididymal/body weight ratio in the high-dose group (Table 1).

GROSS PATHOLOGY (PARENTAL ANIMALS)
No gross abnormality was observed in any organ of treated or control rats at the necropsy examination. One rat in group 2 (250 mg/kg) had a very small testis and was considered an incidental finding not related to treatment. The seminiferous tubules of this small testis were markedly atrophied as denoted by lack of germinal cells and spermatogenesis. Testes, prostate gland, and kidneys were considered the main organs affected by the treatment with the test substance. Eighteen of twenty (90%) rats in the highest dose group (2000 mg/kg) had testicular lesions characterized by lack of spermatogenesis and by degeneration, vacuolization, and necrosis of spermatogonial cells. The hypospermatogenic tubules were reduced in diameter and were lined primarily with Sertoli cells. These affected tubules were located mostly in the sub-capsular region of the testes

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic changes were observed in the prostate glands of 1 of 20 (5%) rats in 1000 mg/kg and 4 of 20 (20%) in 2000 m/kg dose groups. These changes were characterized by multifocal infiltration of lymphocytes and plasma cells into the interstitium, and dilatation of acini containing cellular debris (3 rats in 2000 m/kg and 1 in 1000 mg/kg dose groups.
The kidneys of treated rats had varying degrees of regeneration, hyaline droplet degeneration, cytoplasmic hyaline bodies, and cellular infiltrate, primarily lymphocytes, into the interstitium. These changes were more obvious and observed more frequently in the 2000 mg/kg group compared with the control. One rat in group 2 (250 mg/kg) developed sperm granuloma in the epididymis; however, similar changes were not observed in the epididymis of control or other treated rats.

OTHER FINDINGS (PARENTAL ANIMALS)
No alterations in plasma LH or FSH levels were seen. The findings on the four kill days were comparable for all parameters measured.

Effect levels (P0)

1

Dose descriptor:

LOAEL

Remarks:

Fertility

Effect level:

250 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: the test substance altered reproductive function at all dose levels, while histologic abnormalities of the testes were seen only in the high-dose group

2

Dose descriptor:

LOAEL

Remarks:

toxicity

Effect level:

2 000 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: see 'Remark'

Results: F1 generation

Details on results (F1)

See above

Effect levels (F1)

Dose descriptor:

other: see above

Generation:

other: see P0

Basis for effect level:

other: see above

Remarks on result:

other: see parental findings

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Body weights and Organ/body weight ratios of male Fischer 344 rats receiving dimethyl phosphonate for 13 weeks

Dose (mg/kg)	Body weight (g; Mean±SE)		Organ to body weight ratio (Mean±SES)
	Initial	Final	Testis	Epididymis	Prostate	Kidney
0	112.0±1.5	288.9±4.3	5.1±0.1	1.5±0.02	1.4±0.06	7.4±0.1
250	111.2±1.4	283.9±4.3	5.2±0.1	1.5±0.02	1.3±0.07	7.1±0.1
500	111.2±1.6	272.9±4.6	5.4±0.1	1.6±0.02	1.2±0.08	7.3±0.1
1000	111.8±1.6	276.0±5.1	5.5±0.1	1.6±0.02	1.3±0.05	7.8±0.1**
2000	111.2±1.6	258.8±6.4**	5.2±0.1	1.4±0.03**	1.3±0.07	8.3±0.1**
All groups had 20 animals; **: P<0.01 vs. control

 

Table 2: Analysis of sperm from male Fischer 344 rats receiving dimethyl methyl phosphonate (DMMP) for 13 weeks

Dose (mg/kg)	Percentage motile sperm (a) (Mean±SEM)	Sperm count (mean±SE) per g Caudal epididymal tissue)	Sperm head abnormalities (Mean±SEM)
0	80.2±2.7	541.4±25.1 x 10E6	4.5±0.3
250	80.5.0±3.0	515.2±38.9 x 10E6	5.6±0.7
500	79.7±3.6	459.2±35.2 x 10E6	5.9±0.5
1000	71.5.0±3.1*	532.2±38.5 x 10E6	6.9±0.7
2000	35.8±5.5**	219.6±34.0 x 10E6**	41.7±5.1**
All groups had 20 animals; **: P<0.01 vs. control

 

Table 3: Reproductive function of male Fischer 344 rats receiving dimethyl methyl phosphonate for 13 weeks

Dose (mg/kg)	Percentage of males with sper-positive females(a)	Male fertility index(b) (%)	Number of males impregnating 0, 1 or 2 females			Total number (%) of pregnant females(c)	Number of live fetuses per litter (Mean±SEM)	Percentage resorptions(d)
			0	1	2
0	75 (15/20)	70 (14/20)	6	8	6	20 (50)	7.6±0.7	6.1 (10/163)
250	85 (17/20)	75 (15/20)	5	11	4	19 (47.5)	7.8±0.4	14.9 (26/174)*
500	60 (12/20)	60 (12/20)	8	7	5	17 (42.5)	5.7±0.6**	39.4 (63/160)**
1000	50 (10/20)	40 (8/20)	12	5	3	11 (27.5*)	0.82±0.5**	79.1 (34/43)**
2000	55 (11/20)	0**	20	0	0	0 (0.0**)	0.0±0.0**	-
Trend	P<0.05	P<0.01				P<0.01	P<0.01	P<0.01
(a)[(No, of males with 1 or 2 sperm-positive females)/(A' = 20)] X 100; (b)[(No, of males with 1 or 2 pregnant females)/(N=20)] x 100. This value also included pregnant females that did not have sperm-positive smears; (c)Total of 40 females were exposed to 20 males (2 females per male) for each group; (d)[(No, of resorptions)/(total fetuses + total no. of resorptions)] x 100; statistical analysis based on % resorptions per litter; (e)Includes 4 dead fetuses from 2 litters; *: P<0.05 vs. controls; **: P<0.001 vs. controls