Dibutyl phosphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

other: read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Reliability of source suty = R1

Justification for type of information:

Justification for Read Across is provided in Section 13 of IUCLID

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2002

Reference 2

Reference Type:

secondary source

Title:

DIMETHYL PHOSPHONATE CAS N°: 868-85-9

Author:

OECD

Year:

2004

Bibliographic source:

OECD SIDS - UNEP Publication

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Source Chemical 01

IUPAC Name:

Source Chemical 01

Test animals

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.

Administration / exposure

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

TEST ITEM ADMINISTRATION
- Concentrations: 30, 90 or 270 mg/kg bw/day

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:

Female F0 rats were treated 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.sssss
F0 males were each treated 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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

90 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

270 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 male and 12 female rats per dose

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 levels of 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 30, 90, and 270 mg/kg bw/day were selected in the present study.

Positive control:

no

Examinations

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: 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Mortality:

mortality observed, treatment-related

Description (incidence):

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/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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

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

Reproductive function: sperm measures:

not examined

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

Details on results (P0)

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.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general toxicity to males and females

Effect level:

ca. 90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

histopathology: non-neoplastic

2

Dose descriptor:

LOAEL

Remarks:

general toxicity in males and females

Effect level:

270 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

histopathology: non-neoplastic

3

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

histopathology: non-neoplastic

4

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

270 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

270 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

ovary

Treatment related:

yes

Results: F1 generation

General toxicity (F1)

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

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity F1

Generation:

F1

Effect level:

ca. 90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

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

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

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

Any other information on results incl. tables

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.

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. 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 the test item. Insemination and fertility indices were distinctly reduced at 270 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:

NOAEL (development, fertility, general toxicity) = 90 mg/kg bw/day (nominal)

Executive summary:

The reproductive toxicity of the substance was evaluated in an experimental study performed according to the OECD Guideline 421. The test item was administered by oral gavage to groups of 12 male and 12 female Wistar rats at doses of 30, 90 and 270 mg/kg bw/day in polyethylene glycol (PEG 400) from two weeks before mating until 4 to 6 days after parturition (females) or until day 39 of treatment (males).

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. Based on the absence of adverse effects in the 30 and 90 mg/kg bw/day groups and the toxicity of the test item to females in particular at the high dose of 270 mg/kg bw/day, the NOAEL for general toxicity is considered to be 90 mg/kg bw/day.

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 the test item. Insemination and fertility indices were distinctly reduced at 270 mg/kg bw/day.

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. 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, therefore, the subsequent NOAEL for reproduction (fertility and development) was set at 90 mg/kg bw/day.