Methylphosphonic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-23 till 2011-12-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methylphosphonic acid, compound with amidinourea (1:1)

EC Number:

282-758-4

EC Name:

Methylphosphonic acid, compound with amidinourea (1:1)

Cas Number:

84402-58-4

Molecular formula:

C2H6N4O.CH5O3P

IUPAC Name:

methylphosphonic acid, compound with amidinourea (1:1)

Constituent 2

Reference substance name:

Phosphonic acid, methyl-, compound with (aminoiminomethyl) urea (1:1)

IUPAC Name:

Phosphonic acid, methyl-, compound with (aminoiminomethyl) urea (1:1)

Details on test material:

technical product

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Ninety time-mated female New Zealand White rabbits of 4-5 months were obtained from a colony maintained under SPF conditions at Centre Lago, Vonnas, France. The time-mated females arrived at the testing facilities on GD 1 (23-25 November 2011 for Lot 1, 2 and 3, respectively). The rabbits were checked for overt signs of ill health and abnormalities upon arrival.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content, homogeneity and stability of the test substance in the carrier (tap water) were confirmed by HPLC-UV analysis.
Dilutions of the test substance in the vehicle were prepared weekly (namely on 25 November, 2 December, 5 December (only the adapted high-dose level), 9 December and 16 December 2011), and used within 9 days after preparation. The dilutions were stored in a refrigerator (2-10°C) in portions sufficient for one day. The vehicle for dosing the controls was similarly stored.

Details on mating procedure:

Ninety time-mated female New Zealand White rabbits of 4-5 months were obtained from a colony maintained under SPF conditions at Centre Lago, Vonnas, France. The time-mated females arrived at the testing facilities on GD 1 (23-25 November 2011 for Lot 1, 2 and 3, respectively).

Duration of treatment / exposure:

22 consecutive days
The test substance was administered once daily by oral gavage, as a dilution in tap water from GD 6 up to and including GD 28. Controls were treated with vehicle (tap water) only. The animals were dosed for the last time on the day prior to necropsy.

Frequency of treatment:

once daily

Duration of test:

Arrival of time-mated animals : 23-25 November 2011 (on GD 1)2
Start of the treatment : 28-30 November 2011 (from GD 6)
Date of scheduled necropsy : 21-23 December 2011 (GD 29)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 300, 500/400 mg MPAAU/kg bw/day
Basis:
analytical conc.

No. of animals per sex per dose:

22 pregnant females/group

Details on study design:

On account of adverse effects observed, the high-dose level was reduced to 400 mg MPAAU/kg body weight/day from GD 14.

Examinations

Maternal examinations:

Clinical observations: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon for dead or moribund animals. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
Animals showing signs of severe intoxication, particularly if death appeared imminent, were humanely killed to prevent loss of tissues by autolytic degeneration.

Body weight: The body weight of each animal was recorded on GD 1, 6, 9, 12, 15, 18, 21, 24 and 29.

Feed consumption: The feed consumed by each mated female was measured over the periods: GD 1-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-29 by weighing the feeders.

Water consumption: Water consumption was not measured.

Necropsy: The study was terminated with the necropsy of the rabbits on GD 29 (on 21, 22 and 23 December 2011 for the rabbits of lot 1, 2 and 3, respectively). Two high-dose rabbits, who had an early delivery were necropsied during partus on GD 28¹.
The rabbits were killed by an intravenous injection in the ear of sodium pentobarbital and examined for gross abnormalities. Fetuses were killed by hypothermia. Any dam found dead or killed in extremis during the study was also examined macroscopically, and the number of corpora lutea and implantations were recorded. Maternal tissues showing severe macroscopic abnormalities and the kidneys were sampled and fixed in a neutral, aqueous phosphate buffered 4% solution of formaldehyde for possible future microscopic examination.

¹ Rabbit no 149 (lot 2) had an early delivery and was necropsied (during partus) on 21 December 2011.
Rabbit no 167 (lot 3) had and early delivery and was necropsied (during partus) on 22 December 2011.

Ovaries and uterine content:

The uteri (including the fetuses), ovaries and placentas of all females killed at the end of the study were examined for the following parameters:
- number of corpora lutea
- number of implantation sites
- number of early and late resorptions
- number of live and dead fetuses
- number of grossly visible malformed fetuses and fetuses with external
abnormalities
- weight of ovaries
- weight of uterus, containing placentas and fetuses
- weight of uterus, empty
- weight of fetuses
- weight of the placentas
- gross evaluation of the placentas.

Fetal examinations:

Fetopathological examination:
All fetuses of all litters were examined by careful dissection for visceral abnormalities, but visceral examination of the head was conducted in only half of the fetuses in each litter (the head of the other fetuses was subjected to skeletal evaluation, see below). During the visceral examination, the sex of the fetuses was determined.
To examine the head for visceral abnormalities, half of the number of foetuses in each litter was decapitated and their heads fixed in Bouin’s fixative and subsequently free-hand sectioned according to Van Julsinga and Bennett (1977). The sections obtained were examined for visceral abnormalities.
After the visceral examinations, the intact and decapitated bodies were eviscerated, fixed in 70% alcohol, cleared in potassium hydroxide and stained with Alizarin Red S. All skeletons of each group were examined for skeletal abnormalities and then retained. The skull was examined in only half of the fetuses of each litter (the head of the other fetuses was subjected to visceral evaluation, see above). During the fetopathological examinations the observer was unaware of the group of the fetuses.

Statistics:

Statistical analysis of the results:

The data were analysed using the methods mentioned below.
Clinical findings, mortality data, number of pregnant females, females with live fetuses, parental necropsy observations and the fetopathological screening: Fisher's exact probability test.
Body weight, body weight gain, organ weights and feed consumption data: one way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
Number of corpora lutea, implantations, live and dead embryos or fetuses and early and late resorptions: Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test.
Statistical evaluations on variables associated with the fetuses were considered on a litter basis in accordance to standard procedures. The statistics applied are indicated in the tables.
Results were taken as statistically significant where the probability of the results was <0.05 or <0.01. The final interpretation of results was based not only on statistical analysis but also on other considerations such as nature of the finding, dose-response relationships and whether the results were significant in the light of other biological and pathological findings.

Indices:

Reproductive performance:

For each group the following data were presented:
- number of females mated
- number of females pregnant
- number of females with no viable fetuses
- number of females with live fetuses
- number of corpora lutea
- number of implantation sites
- number of live fetuses
- number of dead fetuses
- number of live male fetuses
- number of live female fetuses
- number of early/late/total number of resorptions.

For each group the following indices were calculated:
- female fecundity index = (number of pregnant females/number of females mated) x 100
- pre-implantation loss = [(number of corpora lutea - number of implantation sites)
/ number of corpora lutea] x 100
- post-implantation loss = [(number of implantation sites- number of live fetuses)
/ number of implantation sites] x 100
- gestation index = (number of females with live fetuses/number of females pregnant) x 100
- sex ratio = (number of live male fetuses/number of live fetuses) x 100.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Summary of parental data
Maternal toxicity were noted in the high-dose group and included:
- Mortality; one rabbit of the high-dose group was found dead on GD 9. Two other rabbits of this group were killed in extremis on GD 13 and GD 21.
- Conditional decline; 10 rabbits in the high-dose group showed blood around the perineum and/or in the cage.
- Weight loss or growth retardation, and reduced feed intake were noted in the high-dose group.
These changes were most evident in the initial phase of the study, but remained after reduction of the high-dose level to 400 mg/kg bw/day.
No treatment-related macroscopic findings of the dams were observed at Caesarean section. The carcass weight was reduced in the high-dose group. Two high-dose rabbits had an early delivery and were necropsied (during partus) on GD 28.
- Feed intake also tended to be reduced in the mid-dose group, but the differences with the controls were not statistically significant.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Reproduction data
A number of 22 mated females per group resulted in 21, 20, 18 and 17 litters for the control, low-dose, mid-dose and high-dose group, respectively. Two high-dose rabbits (no’s 149 and 167) had an early delivery and were necropsied (during partus) on GD 28. Results obtained in these two rabbits are not included in the mean data (tables), but all data are shown in the individual data (appendices).
The percentage of live fetuses per animal was statistically significantly decreased in the high-dose group. Postimplantation loss and the number of resorptions per animal were increased in this group. The latter finding was statistically significant and mainly due to an increase in late resorptions.

Fetal placental findings
Fetal placental findings were not affected by the treatment.

Fetal and placental weights
Fetal weights were decreased in the mid- and high-dose group. The differences with the controls were statistically significant for all fetuses and for male fetuses. Fetal weights were also relatively low in the low-dose group. The latter finding was not statistically significant, and, moreover, due to the higher number of fetuses per animal in this group. Therefore the changes in fetal weights in the low-dose group are not considered to be treatment-related.
Placental weights were not affected by the treatment.

Fetal external observations
There was one fetus in the mid-dose group (from dam no.105) showing a malformed head (namely agenesis of all soft tissues and all skull bones, except tongue and lower jaw). One fetus, from a high-dose dam (no. 149) with an early delivery, showed absence of the cranial vault. Umbilical hernia was observed in one fetus of the low-dose and the mid-dose group, and a small fetus was noted in the low-dose and the high-dose group.
Because there was no dose-response relationship, the latter findings were not ascribed to treatment.

Visceral examination

Visceral malformations
One fetus in the mid-dose group (from dam no.105) showed agenesis of all soft tissues and all skull bones, except tongue and lower jaw . The umbilical hernia, observed in one fetus of the low-dose and the mid-dose group was associated with lobular torsion of the liver. Retrocaval uterus was an incidental finding noted in two control fetuses and one low-dose fetus. No other visceral malformations were observed.

Visceral anomalies
No statistically significant or treatment-related differences in the incidences of visceral anomalies were observed among the groups.

Visceral variations
The incidences of fetuses and litters showing a lobular appendix of the gallbladder were statistically significantly increased in the high-dose group.
Four fetuses of the high-dose group showed cysts in the ovaries. This finding was statistically significant, but because it involved only one litter it is probably incidental. Other incidental findings were statistically significant decreased incidences of haemorrhagic areas in the uterus (low- and mid-dose group), and haemorrhagic fluid in the pericard and abdomen (high-dose group). No other statistically significant visceral variations were observed.

Skeletal examination

Skeletal malformations
One fetus in the mid-dose group (from dam no.105) showed agenesis of all skull bones and all soft tissues, except lower jaw and tongue. One fetus, from a high-dose dam with an early delivery (no. 149), showed agenesis of frontal, parietal and supra occipital skull bones. Acephaly has been observed, though at a low incidence, in previous studies conducted in our laboratory with this strain of rabbits without a correlation with treatment. Because of the absence of similar or correlated findings in any other animal in this study, the present findings are not ascribed to treatment.
Other skeletal malformations included the ribs, sternebrae, vertibral column, thoracal bodies and arches. They were equally divided among the various groups and/or present in one or a few fetuses. Because no remarkable or statistically significant differences between the treatment groups and the controls were observed in the incidences of these findings, they were not ascribed to treatment.

Skeletal anomalies
No statistically significant differences between the treatment groups and the controls were observed in the incidences of skeletal anomalies.

Skeletal variations
The incidence of fetuses showing an irregular shape of one strenebra was slightly, though statistically significantly increased in the high-dose group. This finding is probably a chance finding because the incidence of irregular shape of ‘two or more’ strenebrae was not affected.
No other statistically significant differences between the treatment groups and the controls were observed, except for an incidental decrease in fetuses showing accessory lumbar rib(s) in the mid-dose group.

Skeletal retardations
Various statistically significant differences were observed in incidences of retardations in ossification, namely:
- Sternebrae: The incidence of fetuses showing one unossified sternebra was increased in the high-dose group.
- Coracoids: The incidence of fetuses showing unossified coracoid was increased in the mid- and high-dose group. An incidental decrease was noted in incidence of incompletely ossified coracoid in the low-dose group.
- Metacarpals: The incidence of fetuses showing 1-2 unossified metacarpals was increased in the mid-dose group, but this finding was not confirmed in the high-dose group.
- Phalanges: The incidence of fetuses showing 1-4 incompletely ossified medial digits of the frontal phalanges was increased in the high-dose group.
In the low-dose group, the incidence of fetuses with 1-4 unossified medial digits of the frontal phalanges was increased. This finding was, however, not confirmed by dose-related or statistically significant changes in the higher-dose groups and is therefore considered a chance finding.
- Epiphyses of the femur: The incidence of fetuses of which the distal epiphysis of the femur was unossified was increased in the mid- and high-dose group.
- Epiphysis of the tibia: The incidence of fetuses of which the proximal epiphysis of the tibia was unossified was increased in the mid- and high-dose group.
An incidental decrease in incompletely ossified proximal epiphysis of the tibia was observed in the fetuses of the low-dose group.
- Epiphysis of the humerus: The incidence of fetuses of which the proximal epiphysis of the humerus was unossified was increased in the mid- and high-dose group. The incidence of fetuses with the distal epiphysis of the humerus unossified, was increased in the low- and mid-dose group. This finding was, however, not confirmed by dose-related or statistically significant changes in the high-dose group and is therefore considered a chance finding.
As a result of the above retardations in ossification, the total incidence of fetuses showing skeletal retardations was increased in the mid- and high-dose group.

Summary of reproduction data
- No differences were noted in the female fecundity and gestation indices. No effects on the number of corpora lutea, implantation sites, live and dead fetuses, sex ratio, placenta or placental weights were observed.
- The gravid uterus weight tended to be lower in the high-dose group.
- The percentage of live fetuses per animal was decreased and postimplantation loss and the number of (late) resorptions per animal were increased in the high-dose group.
- Fetal weights were decreased in the mid- and high-dose group.

Summary of fetal examination
One fetus of a high-dose dam that had an early delivery showed absence of the cranial vault (agenesis of frontal, parietal and supra occipital skull bones). A fetus of a mid-dose dam showed a malformed head (agenesis of all soft tissues and all skull bones, except tongue and lower jaw). These findings were considered incidental and not ascribed to treatment. There were no other remarkable visceral and skeletal malformations or anomalies. However, a visceral variation (increased incidence of a lobular appendix of the gall bladder) was noted in the high-dose group, while several skeletal retardations in ossification were observed in the mid- and the high-dose group.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

MPAAU was not teratogentic in the NZW rabbit. The developmental no-observed-adverse-effect level (NOAEL) was 500 mg MPAAU/kg body weight/day.

Executive summary:

The objective of this study was to provide data on the effects of MPAAU on pregnant New Zealand White rabbits and the development of the embryo and fetus following daily oral administration by gavage during gestation days (GD) 6-28. The dose levels were 0 (tap water only), 50, 300 or 500 mg MPAAU/kg body weight/day. On account of adverse effects observed, the high-dose level was reduced to 400 mg MPAAU/kg body weight/day from GD 14. The dams were sacrificed on GD 29 and macroscopically examined. Fetuses, placentas and reproductive organs were weighed. The fetuses were macroscopically examined and processed for visceral and skeletal examinations.

Marked maternal toxicity were observed in the high-dose group, and the maternal no-observed-adverse-effect level (NOAEL) was 300 mg MPAAU/kg body weight/day.

No visceral or skeletal malformations or anomalies were observed at any dose level that are treatment related. Effects on reproductive parameters were noted in the high-dose group, consisting of  increased postimplantation loss and decreased percentage of live fetuses per animal. A visceral variation was noted in the high dose group. In the mid- and high-dose group, fetal weights were decreased and skeletal retardations in ossification were observed.

No frank malformation were observed. The visceral variation in the high dose group, and the skeletal retardations in ossification in high and mid dose groups are considered transient in nature and deemed to be secondary to reduced maternal feed intake and the overall growth retardation in these groups. Therefore, the results indicate that MPAAU was not teratogentic in the NZW rabbit. The developmental no-observed-adverse-effect level (NOAEL) was 500 mg MPAAU/kg body weight/day.