Dimethyl methylphosphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: scientifically acceptable study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Determination of teratogenicity of test substance after treatment of female rats from gestation day 6 to gestation day 15

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): 80 021/B
No additional data

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: the albino rats used in the present study were Sprague-Dawley derived (Tif/RAI) and obtained from a closed SPF breeding colony
- Age at study initiation: 2 months at the start of the study
- Weight at study initiation: means of 200 g (females)
- Housing: throughout the experiment the successfully mated were kept in groups of 5 in Macrolon cages in an air-conditioned room at a temperature
- Diet (e.g. ad libitum): the standard diet fed was Nafag No.890
- Water (e.g. ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±0.5°C
- Humidity (%): 60±5%
- Photoperiod (hrs dark / hrs light): the room was illuminated for 10 hours daily
No additional data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 5, 50, 100 and 125 mg/ml for the respective dose levels (100, 1000, 2000 and 2500 mg/kg)
- Amount of vehicle (if gavage): the amount of fluid administered was 20 ml/kg of body weight
- Lot/batch no. (if required): Prod. No.8322, from own stocks
- Purity: a 2% solution of a sodiiim carboxymethylcellulose was used as vehicle

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/3
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no; only male with proven fertility were used
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from day 6 until day 15 of pregnancy (with the exception of the high dose group of the supplementary study, treated day only during the organogenesis from day 6 to day 10 of pregnancy; see below)

Frequency of treatment:

daily

Duration of test:

the dams were killed at day 21 of pregnancy

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 1000 and 2000 mg/kg bw (first experiment; main study); 0, 2000 and 2500 mg/kg bw (second experiment; supplementary study);
Basis:
actual ingested

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The experiment was conducted in two parts, a main study comprising a no effect dose and toxic dose levels, and a supplementary study (further evaluation of possible effect of the compound on embryonic and foetal development) in which the effects of treatment at very high doses was examined in particular.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: see below

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: general condition and symptomatology were checked daily

BODY WEIGHT: Yes
- Time schedule for examinations: weight gain was checked daily

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; food consumption was noted on Days 6, 11, 16 and 21 of pregnancy

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: dams were killed by dry ice and fetuses removed by Caesarean section on day 21 of pregnancy
- Organs examined: following assessment of the dams organs, especially of the ovaries and uterus (mucosa and contents, including amniotic fluid and placentae as well as abortions and resorption sites).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: Yes: No data

Statistics:

t-test (no additional detail provided

Indices:

yes (see Table 1)

Historical control data:

were available

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
DETAILED CLINICAL OBSERVATIONS
- Main study: no test substance dependant adverse effects reported

- Supplementary study: no test substance dependant adverse effects reported

BODY WEIGHT
- Main study: at the 2000 mg/kg dose level dams displayed a marked reduction in body-weight gain throughout the period of treatment.

- Supplementary study: in both the dose groups feed intake and body weight were diminished during the period of treatment

FOOD CONSUMPTION
- Main study: at the 2000 mg/kg dose level dams displayed a marked reduction in feed intake throughout the period of treatment. The dams of the 1000 mg/kg dose solely showed a slight decrease in feed intake.

- Supplementary study: in both the dose groups feed intake was diminished during the period of treatment

Effect levels (maternal animals)

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
CLINICAL SIGNS MORTALITY (Table 1)
- Main study: the rates of either implantation or embryo-lethality (resorptions) were comparable for all groups.
The average weight of the live fetuses was significantly diminished in the 1000 mg/kg and 2000 mg/kg dose groups.

- Supplementary study: the rates of either implantation or embryo-lethality (resorptions) were comparable for the three experimental groups, including the vehicle control.
The average weight of the fetuses was significantly reduced in both the dose groups.

SOFT TISSUE EXAMINATIONS
- Main study: In two fetuses of the 2000 mg/kg dose group slight edema-like changes of the subcutaneous tissue (anasarca) were detected by slicing. Two fetuses showed hypoplasia of lungs in association with dystopia cordis. The incidences concerning hydrocephaly as well as hypoplasia of lungs and anasarca were found to be not beyond the 99 % confidence limits of the vehicle control. Both, hypoplasia of lungs and anasarca, are the most frequent anomalies occurring in untreated controls.

- Supplementary study: the visceral examination of fetuses by applying the slicing technique revealed the occurrence of an internal hydrocephalus in one fetus of the 2500 mg/kg dose group. In a further fetus from another litter of this dose group hypoplasia of lungs was detected. Slight edema-like change of subcutaneous tissue was observed in one fetus of the vehicle control. Spontaneous anomalies have occurred in the cumulative of untreated controls too.

SKELETAL EXAMINATIONS (Table 1)
- Main study: The skeletal assessment did not reveal any significant deviation from the vehicle control in the 100 mg/kg dose group. In both the higher dose groups, however, skeletal maturation in the fetuses was found to be reduced; the phalangeal nuclei of fore and hind limb, the calcaneous and the 5th sternebra were affected in this respect. The incidences concerning the few skeletal anomalies observed among the fetuses of the high-dose group and listed in Table 6 are still ranging within the 99 % confidence limits of the vehicle control.

- Supplementary study: skeletal assessment revealed in both the dose groups an increase in the numbers of still unossified phalangeal nuclei of fore and hind limb and calcaneus. In the 2500 mg/kg dose group, in addition, there was an increased number of still incompletely ossified 5th stemebrae. One instance of irregularly-shaped stemebrae was detected in either the 200 mg/kg dose group or the 2500 mg/kg dose group.

HEAD EXAMINATIONS
- Main study: six fetuses from one litter of the 2000 mg/kg dose group showed slight edema of the cervical region by gross inspection. One out the 302 live fetuses of this high dose group appeared to be suspicious with regard to hydrocephaly. By applying the slicing technique an internal hydrocephalus was found in this fetus.

- Supplementary study: one fetus of the 2'500 mg/kg dose group showed hypoplasia of the lower jaw. This type of malformation was also found among two out of three mal-developed fetuses of the cumulative control

Effect levels (fetuses)

1

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: fetotoxicity

2

Dose descriptor:

NOAEL

Effect level:

> 2 500 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Fetus parameters at indicated experiment and dose level

Parameters	Cumulative historic control	Main study				Supplementary study
		0 mg/kg	100 mg/kg	1000 mg/kg	2000 mg/kg	0 mg/kg	2000 mg/kg	2500 mg/kg
Number of dams	570	25	25	25	25	25	25	25
Spontaneous dam deaths	0	0	0	0	0	1	0	0
Number of dam with deciduomata	0	0	0	0	0	0	0	1/25
Number of dams with implantations	508(a)	23	22	21	23	22/24	24/25	24/25
Mean number of implantations (%)	13.56	13.04	12.45	12.10	14.39	13.1±1.6	13.9±1.2	13.5±1.7
Embryonic resorptions (%)	7.1	9.0	9.5	5.1	7.0	4.7	9.9	9.9
Fetal resorptions (%)	0.1	0	0	0	0.9	0.4	0.3	0
Dead fetuses (%)	0.1	0	0	0	0.9	0.4	0.3	0
Live fetuses (%)	92.5	91.0	90.5	94.9	91.2	51.1	51.8	49.0
Live fetuses with malformations	8 / 6361(b)	0 / 273	0 / 248	0 / 241	1 / 302(c)	0 / 262	0 / 299	2 / 252(e)
Weight of live fetuses (g)	5.25±0.43	5.38±0.47	5.34±0.40	5.04±0.37(d)	4.02±0.56(d)	5.36±0.45	5.02±0.31(f)	5.15±0.40(f)
(a) One totally aborted litter included; (b) 1 Cleft palate + mandibular hypoplasia, 2 Mandibular hypoplasias, 3 Generalized oedemas and 3 "open eyes"; (c) Hydrocephalus internus (as stated by applying the slicing technique); (d) Significantly reduced when compared with the vehicle control (t test, p < 0.01); (e) Mandibular hypoplasia and Internal hydrocephaly (stated by applying the slicing technique); (f) Significantly lower than in the control group (t test, p<0.01)