Dimethyl phosphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

-In a screening study on rats according to OECD TG 421 (gavage study), effects on fertility were seen in females at 270 mg/kg bw/day in the presence of severe general toxicity (decrease in number of females with corpora lutea and implantation sites) (NOAEL reproduction toxicity: 90 mg/kg bw/day; NOAEL general toxicity: 90 mg/kg bw/day).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

food intake was recorded only during the premating period

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
-Age at study initiation: (P) 12 wks
- Weight at study initiation: (P) Males: 313-345 g; Females: 193-216 g.
- Housing: rats were housed singly under conventional conditions in Makrolon Type IIa cages. During the mating periods females were co-housed overnight with their males in type IIIh 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 CET (artificial illumination: approx. 250 lux, for work in the room: approx. 450 lux). From 17:00 to 05:00 CET orientation light, approx 3-5 lux.

Occasional deviations from these standards occurred, e.g. during cleaning of the animal room. These did not have any apparent influence on the outcome of the study.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

Polyethylene glycol 400 (PEG 400/Lutrol ®)

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

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.

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).

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.

Remarks:

Doses / Concentrations:
0, 30, 90, 270 mg/kg body weight/day
Basis:
actual ingested

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.

Positive control:

No

Parental animals: Observations and 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 male and female animals (only clinical findings were recorded individually).
A detailed clinical observation was performed once a week in males (up to necropsy) and females (up to birth, at birth an on day 4 of lactation).

Body weights: Male F0 rats were weighed prior to the study and thereafter weekly and on the day of their necropsy. 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 in both genders. The feed consumption was determined based on the differences in weight of feed provided and feed which remained unconsumed.

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

Oestrous cyclicity (parental animals):

The following female specific data were recorded and evaluated for all female rats assigned to the present study during the study or at the time of necropsy:
-Duration of gestation
-Livebirth index [%]: Number of viable pups at birth x 100/Number of pups born
-Course of birth
-Number of corpora lutea in the right and left ovary
-Number of implantation sites by staining with 10% ammoniumsulfide
The following organs were investigated histopathologically: ovaries (all groups)

Sperm parameters (parental animals):

The following male specific data were recorded and evaluated in the present study at the time of necropsy: testicular and epididymal weight (left and right testis/epididymis individually).

Litter observations:

The following parameters were investigated and assessed for the F1 pups at birth and up to day 4 to 6 post partum:
-Appearance (including externally visible malformations), general behaviour and mortality: twice daily (once at the weekends, on public holidays and on the day of sacrifice). Only clinical findings were documented by individual animal.
-A detailed clinical observation was done on the day of birth and on day 4 post partum.
-Sex ratio of the pups at birth.
-Individual pup weights at birth (day 0 post partum) and on day 4 post partum.

Postmortem examinations (parental animals):

Scheduled necropsies: Males on day 37 and 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: testes and epididymes in Davidson´solution for 3 to 5 days before transferred to 10% neutral buffered formalin solution.
Prostate, seminal vesicles with coagulation glands, uterus with cervix, vagina, ovaries with oviducts, stomach, esophagus, mamma with skin and gross lesions and physical identifiers in 10% neutral buffered formalin solution.

Postmortem examinations (offspring):

The pups were killed by carbon dioxide asphyxia on day 4 to 6 post partum.

Statistics:

a) Analysis of variance (ANOVA) and in case of signifcant results 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) 2 by 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 preformed 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.

Reproductive 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 implantation sites.

Offspring viability indices:

live birth index [%]: number of viable pups at birth x 100/ number of pups born.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not specified

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

at 270 mg/kg bw : Effects were secondary to the severe maternal toxicity in this group

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

at 270 mg/kg bw : Effects were secondary to the severe maternal toxicity in this group

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

at 270 mg/kg bw : Effects were secondary to the severe maternal toxicity in this group

APPEARANCE, BEHAVIOUR AND MORTALITY OF F0 RATS
Up to the dose of the clinical symptoms occurred with remarkable incidences. At 90 mg/kg bw/day soft feces 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.

One control male had to be killed since of poor general condition. This animal exhibited discolorations in the forestomach and thymus as well as little collapsed lungs possibly due to a misapplication.
Two 270 mg/kg bw /day males were found dead. Necropsy of these males revealed a discoloured liver and/or lungs and showed a clear fluid in the body cavity.
All 270 mg/kg bw/da 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 PRE-MATING 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 2.

WEIGHT OF TESTES AND EPIDIDYMIDES
Individual weight of the testes and epididymides were determined in male animals at necropsy.
At 270 mg/kg bw /day relative testis weights were increased and absolute epididymis weights were significantly decreased (each = 0.01)

GROSS PATHOLOGICAL FINDINGS
At scheduled necropsies more 270 mg/kg bw/day males showed a thickened urinary bladder wall.

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.

TIME TO INSEMINATION
Determination of the time to insemination revealed no treatment-related effect.

DURATION OF GESTATION
The data on gestation length do not indicate any treatment 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 at dose levels up to 90 mg/kg bw/day.

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 effect on lactation behaviour up to 90 mg/kg bw/day.

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.

Dose descriptor:

NOEL

Remarks:

General toxicity in males and females

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Mortality and body weight development were not affected up to the dose 90 mg/kg bw/day

Dose descriptor:

LOEL

Remarks:

General toxicity in males and females

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Remarks:

Reproduction/Developmental Toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

LOEL

Remarks:

Reproduction/Developmental Toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Description (incidence and severity):

up tp 90 mg/kg bw/day

Mortality / viability:

no mortality observed

Description (incidence and severity):

up tp 90 mg/kg bw/day

Body weight and weight changes:

no effects observed

Description (incidence and severity):

up tp 90 mg/kg bw/day

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

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.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: In all prenatal, birth and postnatal parameters of F1 pups were unaffected up to 90 mg/kg bw/day

Reproductive effects observed:

yes

Lowest effective dose / conc.:

270 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Reproductive parameters of male and female F0 animals

Since all 270 mg/kg bw/d 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/d 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/d 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. Final body weight and mean weight of testes and epididymes [g]

DOSE (mg/kg bw/day)	0	30	90	270
Final Body Weight	378.5	389.0	388.3	306.5**
Absolute Testis Weight right	1.7084	1.7444	1.6540	1.5974
Absolute Testis Weight left	1.6630	1.7276	1.6586	1.5878
Absolute Testes Weight	3.3714	3.4720	3.3126	3.1852
Relative Testis weight right	0.4516	0.4494	0.4267	0.5224**
Relative Testis weight left	0.4393	0.4452	0.4277	0.5193**
Relative Testes Weight	0.8909	0.8946	0.8544	1.0417**
Absolute Epididymis Weight right	0.7134	0.7169	0.6908	0.5475**
Absolute Epididymis Weight left	0.6871	0.6969	0.6793	0.5474**
Absolute Epididymides Weight	1.4005	1.4138	1.3701	1.0949**
Relative Epididymis weight right	0.1888	0.1854	0.1780	0.1791
Relative Epididymis weight left	0.1819	0.1801	0.1750	0.1789
Relative Epididymides Weight	0.3707	0.3655	0.3530	0.3580

* statistically significantly different from control, p< 0.05

** statistically significantly different from control, p<0.01

Table 2. 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

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

Table 3. 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 4. Duration of gestation [days]

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

Table 5. 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 Sites per litter	11.00	11.33	12.91	$
Prenatal Loss per 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/d.

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/d.

Table 6. 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 Sites per litter	10.78	11.55	11.91	no data
Prenatal Loss per litter	10.78	11.45	11.73	no data

Table 7. 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 8. 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 9. Mean Body Weight on the F1 Pups [g]

DOSE (mg/kg bw/d)	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

Executive summary:

Eiben E (2002)

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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

90 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-Guideline study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Effects of dimethyl phosphonate on fertility and fetal development were assessed in a screening study according to OECD TG 421 ( (Eiben R and Hartmann E, 2002). This study was selected as key study because it was conducted according to OECD guideline 421 and GLP conditions. Groups of 12 male and female Wistar rats were dosed with 0, 30, 90 or 270 mg/kg bw/day by gavage. F0-animals were treated from 2 weeks before mating to the end of gestation and up to 4 to 5 days of lactation. Males were killed on day 37. Females and pups were killed on days 4 to 5 post partum. Ovaries, testes, epididymides and macroscopically altered tissues of F0 animals were examined histologically. Parameters of general toxicity and fertility, as well as pre- and post-natal development were recorded. In the high dose group (270 mg/kg bw/day), animals of both sexes exhibited clear clinical signs of systemic toxicity (poor general state, apathy, high stepping gait, squatting position, bloody muzzle, piloerection, emaciation, tremor and/or desiccation of skin) and severe body weight loss. Two males of this group were found dead with discolored liver and/or lungs, and all females of this group had to be killed in moribund condition during mating or gestation. At 90 mg/kg bw/day soft feces and/or diarrhea were noted in both sexes more frequently than in the control group. No substance related effects were found at 30 mg/kg bw/day. The NOAEL for parental toxicity in rats (males, females) was set at 90 mg/kg bw/day by the study authors.

Effects on fertility

At 270 mg/kg bw/day the relative testis weight was increased and the absolute epididymidis weight decreased. No pathological changes were found at macroscopic and microscopic examinations in the testis and epididymides. The number of females with corpora lutea and implantation sites was decreased at 270 mg/kg bw/day, as well as the frequency and severity score of “large corpora lutea” and of granular luteal cells. All changes at 270 mg/kg bw/day were considered secondary to the toxic effects of dimethyl phosphonate. Insemination and fertility indices were distinctly reduced at 270 mg/kg bw/day. Reproductive parameters (insemination parameters, fertility index, gestation indices, gestation length, prenatal loss, number of implantation sites, macroscopically visible corpora lutea, life birth index, sex ratio, pup birth weight, litter size, pup weight development, viability and lactation of F1 rats) were not affected at 30 and 90 mg/kg bw/day.

In another reliable study testicular atrophy was observed in mice treated by gavage with 375 mg/kg bw/day for 13 weeks. Probably this effect was secondary to general toxicity. In fact, dimethyl phosphonate at this dose level also induced mortality (NTP 1985). Furthermore, focal calcification in the testis was seen in 9/47 mice dosed with 100 mg/kg bw/day and in 24/50 mice dosed with 200 mg/kg bw/day following chronic exposure (103 weeks). The shape and location of the deposits in the testis suggest mineralization of seminiferous tubules (NTP, 1985). Hypospermatogenesis was reported in rats after inhalation of 483.1 mg/m³ (corresponding to about 100 mg/kg bw/d) for 4 weeks (Mobil Oil Corporation, 1982) in a low quality and therefore not reliable study. These effects were probably secondary to the general toxicity.

Overall, The NOAEL for reproduction toxicity in rats (males, females) was at 90 mg/kg bw/day.

Short description of key information:
-In a screening study on rats according to OECD TG 421 (gavage study), effects on fertility were seen in females at 270 mg/kg bw/day in the presence of severe general toxicity (decrease in number of females with corpora lutea and implantation sites) (NOAEL reproduction toxicity: 90 mg/kg bw/day; NOAEL general toxicity: 90 mg/kg bw/day).
-OECD guideline 408: oral exposure: NOAEL (male/female) =190 mg/kg bw/day, OECD guideline 408; 6 concentrations (0, 95, 190, 375, 750, 1500 mg/kg bw/day) orally administered to mouse. Testicular atrophy, characterized by hypospermatogenesis with the formation of large giant spermatids and syncytial cells, was seen in male mice at 375, 750, and 1500 mg/kg bw/day. These effects were probably secondary to the general toxicity.

Effects on developmental toxicity

Description of key information

No developmental toxicity was found in rats at doses of 30 and 90 mg/kg bw/day (NOAEL developmental toxicity: 90 mg/kg bw/day) in an OECD TG 421 (Reproduction/ Developmental Toxicity screening test).

Link to relevant study records

Reference

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

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

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

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.

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.

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.

Dose descriptor:

NOEL

Effect level:

90 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

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.

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.

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.

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

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

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

90 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-Guideline study.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the afore described OECD Screening Study (Eiben R and Hartmann E, 2002), the sex ratio, mortality and weights of F1 pups were not affected by treatment up to and including 90 mg/kg bw/day, while evaluation was not possible at higher doses as there were no surviving pups. No externally malformed pups were observed.

Toxicity to reproduction: other studies

Description of key information

-OECD guideline 408 (NTP 1985): oral exposure: NOAEL (male/female) =190 mg/kg bw/day, OECD guideline 408; 6 concentrations (0, 95, 190, 375, 750, 1500 mg/kg bw/day) orally administered to mouse. Testicular atrophy, characterized by hypospermatogenesis with the formation of large giant spermatids and syncytial cells, was seen in male mice at 375, 750, and 1500 mg/kg bw/day. These effects were probably secondary to the general toxicity.

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction: other studies

Remarks:

OECD 408 (repeated 90 day oral toxicity in rodents)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remarks'

Remarks:

Comparable to guideline study (No organ weight determination, no haematological/biochemical examinations, no data on statistics). Adopted according to OECD SIDS (public available peer reviewed source). The original source is available and has been reviewed.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 408 (repeated 90 day oral toxicity in rodents)

Deviations:

yes

Remarks:

No organ weight determination, no haematological/biochemical examinations, no data on statistics.

Principles of method if other than guideline:

Necropsy performed on all animals; the following tissues from vehicle control and all but the 95 mg/kg bw dosed group of mice were microscopically examined: gross lesions, skin , parathyroids, colon, esophagus, brain, sternebrae (including marrow), liver, lung and mainstem bronchi, stomach, thymus, pancreas, kidney, urinary bladder, eyes, mandibular lymph node, saliva glands, thyroid gland, small intestine, ovaries/ uterus or prostate / testes, heart, trachea, spleen, adrenal glands, pituitary gland, gallbladder, mammary gland. Only heart, liver, and kidney examined for the 95 mg/kg group of mice.

GLP compliance:

not specified

Type of method:

in vivo

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI, USA).
- Age at study initiation: 6-8 weeks old.
- Weight at study initiation: 23-25 g (male), 18-19 g (female).
- Housing: 5 animals per cage (polycarbonate).
- Diet (e.g. ad libitum): Purina Lab Chow meal (St. Louis, MO); available ad libitum.
- Water (e.g. ad libitum): Acidified with HCl (pH 2.5) tap water; available ad libitum.
- Acclimation period: 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24 (max 28).
- Humidity (%): 30-70
- Air changes (per hr): 12-15 room air changes/h.
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of dimethyl hydrogen phosphite were mixed with corn oil. Mixtures were resuspended before dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was chosen because of the potential for chemical hydrolysis in water.
- Concentration in vehicle: 0, 28.53, 57.06, 112.61, 225.22, 450.45 mg/mL
- Amount of vehicle (if gavage): 3.33 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification of doses or concentrations: Analyses for dimethyl hydrogen phosphite in corn oil were performed on every eighth dose mixture to confirm that the correct concentrations were administered to the test animals. The method of analysis involved a methanolic extraction as a purification step and a gas chromatographic assay as a quantification step.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days

Remarks:

Doses / Concentrations:
0, 95, 190, 375, 750, 1500 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: yes (observed twice per day)
- Mortality: yes (observed twice per day)

- Body weight: yes (weeklyl)

- Organ weight: no

- Food consumption: no

- Water consumption: no

- Haematology: no

- Biochemistry: no

- Urinalysis: no

Necropsy performed on all animals; the following tissues from vehicle control and all but the 95 mg/kg bw dosed group of mice were microscopically examined: gross lesions, skin , parathyroids, colon, esophagus, brain, sternebrae (including marrow), liver, lung and mainstem bronchi, stomach, thymus, pancreas, kidney, urinary bladder, eyes, mandibular lymph node, saliva glands, thyroid gland, small intestine, ovaries/ uterus or prostate / testes, heart, trachea, spleen, adrenal glands, pituitary gland, gallbladder, mammary gland. Only heart, liver, and kidney examined for the 95 mg/kg group of mice.

Dose descriptor:

NOAEL

Remarks:

Reproduction Toxicity

Effect level:

190 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Dose descriptor:

LOAEL

Remarks:

Reproduction Toxicity

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Testicular atrophy, characterized by hypospermatogenesis with the formation of large giant spermatids and syncytial cells, was seen in male mice at 375, 750, and 1500 mg/kg. These effects were probably secondary to the general toxicity.

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
All the mice of each sex that received 750 or 1500 mg/kg died during the first 4 weeks. Two of 10 males and 5 of 10 females that received 375 mg/kg also died. Mice that received 375 mg/kg or more had tremors and decreased activity. Final weights of surviving dosed and vehicle control mice were comparable. Lung congestion in males and females, cardiac mineralization in males, and hepatocellular vacuolization in females were probably compound related. Pulmonary congestion was observed in animals that died during the studies. Testicular atrophy, characterized by hypospermatogenesis with the formation of large giant spermatids and syncytial cells, was seen in male mice at 375, 750, and 1500 mg/kg.
These effects were probably secondary to the general toxicity.

Table1. Survival and mean body weights of mice in the thirteen-week gavage studies of dimethyl hydrogen phosphite.

		Mean Body Weights (a) (grams)
Dose (mg/kg)	Survival (b)	Initial	Final	Change (c)	Final Weight Relative to Vehicle Controls (percent)
MALE
0	10/10	24	29	+ 5	--
95	10/10	24	30	+ 6	103.4
190	10/10	25	31	+ 6	106.9
375	(d) 8/10	24	28	+ 4	96.6
750	(e) 0/10	25	(f)	(f)	(f)
1500	(g) 0/10	23	(f)	(f)	(f)
FEMALE
0	10/10	18	23	+5	--
95	10/10	18	23	+5	100.0
190	10/10	18	22	+4	95.7
375	(f) 5/10	19	24	+5	104.3
750	(g) 0/10	18	(f)	(f)	(f)
1500	(h) 0/10	18	(f)	(f)	(f)

(a) Only group weights were taken by laboratory; no individual animal weight data are available.

(b) Number surviving /number in group.

(c) Mean weight change of the group.

(d) Week of death: 11, 12

(e) Week of death : 1, 3, 3, 3, 4, 4, 4, 4, 4, 4

(f) No results are reported due to the 100% mortality in this group.

(g) Week of death: 1, 1, 1, 1, 1, 2, 2, 2, 4, 4

(h) Week of death: 5, 10, 11, 12, 12

(i) Week of death: 3, 4, 4, 4, 4, 4, 4, 4, 4, 4

(j) Week of death: 1, 1, 1, 1, 1, 1 ,1, 1, 3, 4

Table 2. Histopathologic lesions observed in mice in the thirteen-week gavage studies of dimethyl hydrogen phosphite.

Dose (mg/kg)	Hepatocellular Vacuolization (a)	Cardiac Mineralization (minimal severity)	Testicular Atrophy	Lung Congestions
MALE
0	1/10	0/10	0/10	0/10
95	(b) 1/10	0/10	0/10	0/10
190	1/10	9/10	0/10	0/10
375	2/10	3/10	3/10	1/10
(c) 750	2/9	0/10	9/10	7/10
(c) 1500	1/10	1/10	2/10	7/10
FEMALE
0	0/10	1/10	-	0/10
95	0/10	0/10	-	0/10
190	5/10	1/10	-	0/10
375	5/10	2/10	-	4/10
(c) 750	0/9	0/9	-	7/10
(c) 1500	2/7	0/10	-	9/10

(a) Male: diffuse or focal: diffuse.

(b) Observed by quality assurance pathologist.

(c) Most animals in these groups died early.

Executive summary:

A repeated dose toxicity study equivalent or similar to OECD 408 (repeated 90 day oral toxicity in rodents) was conducted. AnNecropsy performed on all animals. The following tissues from vehicle control and all but the 95 mg/kg bw dosed group of mice were microscopically examined: gross lesions, skin , parathyroids, colon, esophagus, brain, sternebrae (including marrow), liver, lung and mainstem bronchi, stomach, thymus, pancreas, kidney, urinary bladder, eyes, mandibular lymph node, saliva glands, thyroid gland, small intestine, ovaries/ uterus or prostate / testes, heart, trachea, spleen, adrenal glands, pituitary gland, gallbladder, mammary gland. Only heart, liver, and kidney examined for the 95 mg/kg group of mice.

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:  

All the mice of each sex that received 750 or 1500 mg/kg died during the first 4 weeks. Two of 10 males and 5 of 10 females that received 375 mg/kg also died. Mice that received 375 mg/kg or more had tremors and decreased activity. Final weights of surviving dosed and vehicle control mice were comparable. Lung congestion in males and females, cardiac mineralization in males, and hepatocellular vacuolization in females were probably compound related. Pulmonary congestion was observed in animals that died during the studies. Testicular atrophy, characterized by hypospermatogenesis with the formation of large giant spermatids and syncytial cells, was seen in male mice at 375, 750, and 1500 mg/kg.

These effects were probably secondary to the general toxicity.

Additional information

In another reliable study testicular atrophy was observed in mice treated by gavage with 375 mg/kg bw/day for 13 weeks. Probably this effect was secondary to general toxicity. In fact, dimethyl phosphonate at this dose level also induced mortality (NTP 1985). Furthermore, focal calcification in the testis was seen in 9/47 mice dosed with 100 mg/kg bw/day and in 24/50 mice dosed with 200 mg/kg bw/day following chronic exposure (103 weeks). The shape and location of the deposits in the testis suggest mineralization of seminiferous tubules (NTP, 1985).

Hypospermatogenesis was reported in rats after inhalation of 483.1 mg/m³(corresponding to about 100 mg/kg bw/d) for 4 weeks (Mobil Oil Corporation, 1982) in a low quality and therefore not reliable study. These effects were probably secondary to the general toxicity.

Justification for classification or non-classification

In the screening study on rats according to OECD TG 421 (gavage study), effects on fertility were seen in females at 270 mg/kg bw/day in the presence of severe general toxicity.

Testicular atrophy was observed in mice treated by gavage with 375 mg/kg bw/day for 13 weeks. Probably this effect was secondary to general toxicity. In fact, dimethyl phosphonate at this dose level also induced mortality (NTP 1985).

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.