Dimethyl phosphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

Reference Type:

secondary source

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 421 (Reproduction/Developmental Toxicity screening test)

Deviations:

yes

Remarks:

food intake was recorded only during the premating period

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl phosphonate

EC Number:

212-783-8

EC Name:

Dimethyl phosphonate

Cas Number:

868-85-9

Molecular formula:

C2H7O3P

IUPAC Name:

dimethyl phosphonate

Details on test material:

- Name of test material (as cited in study report): dimethylphosphit
- Physical state: liquid
- Analytical purity: 99.8%
- Purity test date: 24-10-2001
- Lot/batch No.: 1/030091
- Stability under test conditions: at least 8 days (at animal room conditions)
- Storage condition of test material: at room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Housing: rats were housed singly under conventional conditions in Makrolon Type IIa cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): 10 passages per hour at minimum
- Photoperiod (hrs dark / hrs light): 12 hour rhythm from 05:00 to 17:00

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): PEG 400 was chosen because of the potential for chemical hydrolysis in water
- Concentration in vehicle: 0, 6.0, 18.0, 54.0 mg/mL
- Amount of vehicle (if gavage): 5 mL/ kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical verification of doses and concentration was conducted by capillary gas chromatography (GC).

Details on mating procedure:

Pairing was performed overnight (from about 15:00 to 08:00 CET; on week-ends from 12:00 to 08:00 CET) by placing one F0 female animal together with one F0 male rat into a type IIIh Makrolon cage. Allocation of the female animals to the respective male animals was performed with ascending animals number, i.e. the 1st male of the relevant dose group was paired with the 1st female and so on. During the two-week mating period each female animal was paired daily. Females with positive sperm detection (taken as day 0 of gestation) were not mated again.

F0 females found sperm-positive after the first mating day but were shown to be not pregnant (no increase in weight) were co-housed again (remated) over one week with the same male without checking insemination or measuring body weight.

Duration of treatment / exposure:

Dimethyl phosphonate or vehicle was administered to each of the female F0 rats once daily for 2 weeks prior to mating, during the subsequent mating and remating period, during gestation and up to the day before necropsy (day 4 to 6 post partum) of their pups.
The F0 males were each given dimethyl phosphonate or vehicle once daily for 2 weeks prior to mating, during the following mating up to day 37.

Frequency of treatment:

Daily between 06:00 and 12:30 CET.

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels used were selected according to results of a preceding pilot parental tolerability study with dose level of 0, 30, 100, and 350 mg/kg bw/day. In this study two rats per group and sex were treated two weeks prior mating, as well as mating and lactation period up to 4 day post partum. At 350 mg/kg bw/day all parental rats had to be killed prescheduled because of poor general condition and body weight loss. Necropsies of these rats as well as those of animals, which were necropsied prescheduled, revealed no treatment related gross lesions. In F1 pups no toxic effects were noted up to 100 mg/kg bw/day. From these results it was concluded that the parental toxicity would occur between 100 and 350 mg/kg bw/day. Therefore, the dose levels of 0, 30, 90, and 270 mg/kg bw/day were selected in the present study.

Examinations

Maternal examinations:

Clinical signs: Appearance, behaviour and mortality were monitored twice daily (once daily on weekends, public holidays and on day of necropsy) by cage side examination during the entire treatment period in female animals (only clinical findings were recorded individually).
A detailed clinical observation was performed once a week in females (up to birth, at birth an on day 4 of lactation).

Body weights: F0 females were weighed prior to the study and thereafter weekly. In inseminated females body weights was recorded up to the day of delivery, on day 0 and 4 post partum as well as on the day of their necropsy.

Measurement of food consumption was performed weekly during the premating period. The feed consumption was determined based on the differences in weight of feed provided and feed which remained unconsumed.

Gross pathological examination of all female animals during necropsy on unscheduled or scheduled deaths.

-Determination of insemination day
-Insemination, fertility and gestation index
- Scheduled necropsies: Females on day 4 to 6 post partum of their pups.
All animals were killed by exsanguination under deep carbon dioxide anesthesia.
At necropsy the following organs were fixed: uterus with cervix, vagina, ovaries with oviducts, stomach, esophagus, mamma with skin and gross lesions and physical identifiers in 10% neutral buffered formalin solution.

Ovaries and uterine content:

The uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
-Prenatal Loss per litter: Yes

Fetal examinations:

Not performed.

Statistics:

a) Variance analysis and Dunnett´s test (organ weights, time to insemination, duration of gestation, number of implantation sites per females, prenatal loss per female, live birth and viability index, number of pups delivered, stillborn, died, missing and/or cannibalized, number of live pups per female at the individual weighing times, sex ratio of pups, pup weights and pup weight changes).

b) N CHI² test, in case of significant differences Fisher´s exact test with Bonferroni correction for insemination, fertility and gestation index, number of females with live pups, stillborn pups, all pups stillborn, number of females with total postnatal litter loss up to day 4 post partum.

c) F-test and additional t-test or Welch t-Test (number of implantation sites per female).
These calculations were performed using an HP Vectra personal computer (Basic program) in case there were differences with respect to control group.

d) Dunnett-Test in connection with a variance analysis for body weights of parent animals
e) The Kruskal-Wallis-Test with a Steel-Test for food consumption data.
These calculation were performed using SAS routine on a HP 3000 computer system

Indices:

Insemination index [%]= Number of females inseminated x 100/Number of females paired; Fertility index[ %]=Number of females with implantation sites x 100=Number of females inseminated; Gestation index [%]= Number of females with viable pups x 100/ Number of females with implantations sites; Livebirth index [%]= Number of viable pups at birth x 100= Number of pups born.

Historical control data:

Historical control values taken from fertility studies performed at Bayer AG, from pre-and postnatal studies, and from reproduction/developmental toxicity screening studies.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
APPEARANCE, BEHAVIOUR AND MORTALITY OF F0 RATS
Up to the dose 30 mg/kg bw/day none of the clinical symptoms occurred with remarkable incidences. At 90 mg/kg bw/day soft faeces and/or diarrhoea were noted more frequently in both sexes than at 0 mg/kg bw/day. At 270 mg/kg bw/day several clinical signs of severe toxicity such as poor general condition, apathy, high stepping gait, squatting position, bloody muzzle, piloerection, emaciation, tremor and/or desiccation of the skin were noted in males and /or females.
All 270 mg/kg bw/day females had to be killed in moribund condition during mating or gestation. There were no specific organ changes at necropsy among high dose females.
Taken together there was an increased mortality in both sexes at 270 mg/kg bw/day.

FOOD CONSUMPTION DURING THE PREMATING PERIOD
A toxicologically relevant effect on food intake during the premating period was not evident in males and females at dose level of up to 270 mg/kg bw/day.

BODY WEIGHT DEVELOPMENT
A treatment-related effect on body weight development of males was not evident at dose levels of up to 90 mg/kg bw/day. At 270 mg/kg bw/day there was a severe and increasing body weight reduction from week 2 onwards (p< 0.02) and severe body weight loss beginning with week 3 and with a maximum in week 4.
The mean body weight gain of females was not remarkably changed up to 90 mg/kg bw/day during the premating, mating gestation and lactation period.
At 270 mg/kg bw/day body weight loss was noted in week.

HISTOPATHOLOGICAL INVESTIGATIONS
There was a reduction in frequency and severity score of large corpora lutea as well as of granular luteal cells in 270 mg/kg bw/day females, which all had been necropsied prescheduled.

EFFECTS ON REPRODUCTION OF MALE AND FEMALE F0 ANIMALS
The insemination, fertility, and gestation indices were unchanged up to 90 mg/kg bw/day. At 270 mg/kg bw/day very low insemination and fertility indices were calculated, which was secondary to the severe maternal toxicity in this group. This was the reason that all females of the 270 mg/kg bw /day dose group were sacrificed moribund at the end of the mating period or during early gestation. Therefore, no gestation index could be calculated for this dose and evaluation of data regarding reproductive performance of this dose group was thus limited.

DURATION OF GESTATION
The data on gestation length does not indicate any treatment related effect up to 90 mg/kg bw/day.

COURSE OF BIRTH
The actual process of giving birth could be observed only in a few cases since the animals generally littered at night. Observations indicating a substance-related effect on course of birth were not made of dose levels up to 90 mg/kg.

LACTATION BEHAVIOUR
The was no F1 pup without a visible milk ingestion up to 90 mg/kg bw/day indicating that there was no treatment related effect on lactation behaviour up to 90 mg/kg bw/d.

REPRODUCTION DATA OBTAINED AT NECROPSY
The mean number of macroscopically visible corpora lutea, implantation sites and prenatal loss was not affected up to a dose of 90 mg/kg bw/day. Data received at 270 mg/kg bw/day could not be evaluated due to prescheduled deaths during mating period.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

90 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
POSTNATAL DEVELOPMENT OF THE F1 PUPS
There were no clinical symptoms or necropsy findings in F1 pups, which could be attribute to the treatment up to 90 mg/kg bw/day. Malformed pups were not observed in this study.

LITTER SIZE
The litter size at birth as well the number of pups alive on day 0 and 4 post partum were not affected by treatment at dose levels up to 90 mg/kg bw/day.

MORTALITY OF THE F1 PUPS AND VIABILITY INDEX
No adverse effect could be detected up to 90 mg/kg bw/day.

SEX RATIO OF THE F1 PUPS
Up to the dose 90 mg/kg bw/day no change in the sex ratio was found.

BODY WEIGHT DEVELOPMENT OF THE F1 PUPS
There was no toxic effect on pup weights up to 90 mg/kg bw/day.

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: The sex ratio, mortality and weights of F1 pups were not affected by treatment up to and including 90 mg/kg bw/d, while evaluation was not possible at higher doses as there were no surviving pups. No externally malformed pups were observed.

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: The sex ratio, mortality and weights of F1 pups were not affected by treatment up to and including 90 mg/kg bw/d, while evaluation was not possible at higher doses as there were no surviving pups. No externally malformed pups were observed.

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Any other information on results incl. tables

Reproductive parameters female F0 animals

Since all 270 mg/kg bw/day females had to be killed in moribund state before littering most of the reprotoxicological parameters could not be investigated in this group.

At 30 and 90 mg/kg bw/day insemination parameters, fertility and gestation indices, number of implantation sites, and macroscopically visible corpora lutea as well as gestation lenght were not affected. At 270 mg/kg bw/day the insemination and fertility index were lower than in controls secondary to the severe toxicity. The prenatal loss as well as the number of macroscopically visible corpora lutea and implantation sites could not be evaluated in this group.

Table 1. Insemination Index, Fertility Index and Gestation Index

DOSE (mg/kg bw/ day)	Females Used	Females  Inseminated		Females with Implantations		Females with Viable pups
		n	% of those paired Insemination Index	n	% of those InseminatedFertility Index	n	% of those with ImplantationsGestation Index
0	12	12	100	10	83.3	9	90.0
30	12	12	100	12	100	11	91.7
90	12	12	100	11	91.7	11	100
270	12^1	5^1	41.7	2^1	40.0	0 ^1	0

¹all females sacrificed moribund at the end of the mating period or during early gestation

Table 2. Time to insemination [days] (remated female excluded]

DOSE (mg/kg bw/day)	0	30	90	270
Inseminated females	11	8	8	5
Time to insemination	2.1	2.0	2.1	2.2

Table 3. Duration of gestation [days]

DOSE (mg/kg bw/day)	0	30	90	270
	21.88	21.63	21.88	no data

Table 4. Reproduction Data

DOSE (mg/kg bw/day)	0	30	90	270
No. of Corpora Lutea*all females	10.75	11.67	11.92	$
No. of Corpora Lutea*pregnant female only	12.90	11.67	13.00	$
No. of Implantation Sitesper litter	11.00	11.33	12.91	$
Prenatal Lossper litter	1.20	0.75	1.00	$

* macroscopically visible

$ data could not be evaluated or not availble

Parameters investigated on pups

Prenatal loss, life birth index, pup birth ratio, pup birth weight, overall litter size, pup weight development and viability of F1 rats were unaffected at 30 and 90 mg/kg bw/day.

There were also no remarkable clinical or necropsy findings in pups and no adverse effect on the course of birth or lactation behavior of the dams in these groups.

All in all prenatal, birth and postnatal parameters of F1 pups were unaffected up tp 90 mg/kg bw/day.

Table 5. Litter Size

DOSE (mg/kg bw/day)	0	30	90	270
	Group means per litter
Pups delivered	10.89	11.55	11.91	no data
No. of Implantation Sitesper litter	10.78	11.55	11.91	no data
Prenatal Lossper litter	10.78	11.45	11.73	no data

Table 6. Survival indices of the pups [%]

DOSE (mg/kg bw/day)	0	30	90	270
Live Birth Index	99.26	100	100	no data
Viabilty Index (day 4 p.p)	100	99.30	98.64	no data

Table 7. Sex ratio of the F1 pups [% males per litter]

DOSE (mg/kg bw/day)	0	30	90	270
Day 0 p.p.	54.85	58.72	58.92	no data

Table 8. Mean Body Weight on the F1 Pups [g]

DOSE (mg/kg bw/day)	0	30	90	270
Day 0 p.p.	5.92	5.81	5.73	no data
Day 4 p.p.	9.97	9.33	9.56	no data

Applicant's summary and conclusion

Executive summary:

Groups of 12 male and female Wistar rats each were treated daily orally (by gavage) for two weeks before mating as well as during the mating, gestation and lactation period (up to day 4 or 5 post partum) with dimethyl phosphonate dissolved in Polyethylene glycol 400 in doses of 0, 30, 90 and 270 mg/kg bw/day, respectively. Males were necropsied when have been dosed at least 28 days. Females and their pups were necropsied between day 4 to 6 post partum. Investigations were performed on general tolerance on the test compound by the parental animals including histopathology of testes, epididymides and ovaries as well as with regard to effects on reproduction including early postnatal development of F1 pups. Necropsies were done on all animals. Selected organs of F0 rats were weighed. The study was performed according OECD Guideline No.421 of 1995 with the exception that food intake was recorded only during the premating period. At 270 mg/kg bw/day clinical signs (poor general condition, apathy, high stepping gait, squatting position, bloody muzzle, piloerection, emaciation, tremor and/or desiccation of the skin) and severe body weight depression were found indicating a severe parental toxicity and resulting in increased mortality in males and complete mortality in females. The food intake during the premating period was unaffected up to 270 mg/kg bw/day. At 270 mg/kg bw/day relative testis weights were increased and absolute epididymis weights were decreased secondary to changes in body weight. The frequency and severity score of "large corpora lutea" as well as of granular luteal were reduced in 270 mg/kg bw/day females. These findings are attributed to the effects on the body weights and severe somatic toxicity. Reproductive Parameters At 30 and 90 mg/kg bw/day insemination parameters, fertility and gestation indices, gestation length, prenatal loss, number of implantation sites and macroscopically visible corpora lutea, life birth index, sex ratio, pup birth weight, overall litter size, pup weight development, viability and lactation of F1 rats were not affected. At 270 mg/kg bw/day the reproductive parameters were distinctly reduced secondary, could not be evaluated or could not be measured due to severe somatic toxicity and lethality of the 270 mg/kg bw/day females. No remarkable clinical or necropsy findings in pups occurred in the groups up to 90 mg/kg. Thus, the following no-observed-effect levels (NOEL) were determined: General Toxicity in Males and Females: 90 mg/kg body weight/day Reproduction/Developmental Toxicity: 90 mg/kg body weight/day.