Diphosphoric acid, compd. with piperazine (1:1)

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

457-330-7

EC Name:

-

Cas Number:

66034-17-1

Molecular formula:

Hill formula: C4H10N2:H4O7P2 CAS formula: C4H10N2:H4O7P2

IUPAC Name:

(phosphonooxy)phosphonic acid; piperazine

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Wistar rats, strain: Crl:WI(Han)
- Source: Charles River Deutschland, Sulzfeld
- Age at treatment start: approx. 12 weeks old, both sexes
- Housing in Macrolon plastic cages
during pre-pairing dosing period: In groups of 5 by sex
during pairing: 1 male+1 female/cage
males after pairing: In groups of 5
females during gestation and lactation: Females housed individually (+litter).
- Bedding material (in Macrolon plastic cages): sterilised sawdust as bedding, paper as enrichment and nesting material
- Diet (ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 5 days before treatment start

ENVIRONMENTAL CONDITIONS
The animal room was maintained at (target ranges for temperature and relative humidity):
- Temperature (°C): 18 - 24°C
- Relative Humidity (%): 40 - 70 %
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Ventilation: at least 10 room air changes/h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% aqueous

Details on exposure:

- Amount (dose volume by gavage): 5 mL/kg bw/day.
Actual dose volumes were calculated accounting for the latest bodyweight.

- Frequency of preparation of dose formulations: daily within 6 hours prior to dosing

Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals.

Details on mating procedure:

- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most 14 days, until proof of pregnancy was confirmed. 
- Proof of pregnancy: Formation of vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For analysis of the test substance in the vehicle LC/MS was used with a Hypersil BDS C18 column (100 mm x 4.6 mm i.d., dp = 5 µm). The analytical method was validated. The mean contents of the test substance in dose formulations were found to be well within the acceptance limit (within +/- 15%) of dose theoretical concentration; the relative standard deviation was equal to or less than 10%.

Duration of treatment / exposure:

- Treatment period, parental males: 28 days (14 days before mating, during mating and up to the day prior to scheduled necropsy)
- Treatment period, parental females (dams): 41-54 days (from 14 days prior to mating to at least Lactation Day 4)
- Pups were not treated directly (possibly via milk): until Lactation Day 4.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

Not included in the study.

Examinations

Parental animals: Observations and examinations:

Clinical observations performed and frequency:
- Clinical signs: Daily (covering external appearance, motor activity & morbidity in each animal)
- Body weight, Males: Weekly from Treatment Day 0 to 28.
Body weight, Females: Weekly for pre-pairing & pairing period, Gestation Days 0, 4, 7, 11, 14, 17, 20 & Lactation Days 1 and 4
- Food consumption, Males: Weekly
Food consumption, Females: Weekly for pre-pairing period and for Gestation Days 0, 4, 7, 11, 14, 17 and 20and on Lactation Days 1 and 4

Oestrous cyclicity (parental animals):

not observed

Sperm parameters (parental animals):

not observed

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- Males: Killed on the day after the 28-day treatment period.
- Terminal sacrifice, Females (dams): Killed on Lactation Day 5-7

Gross pathology: Necropsy with tissue collection.
The number of implantation sites and corpora lutea was recorded for all dams

Organs Weights: The following organs were weighed at necropsy : testes, epididymides.

Histopathology: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
testes, epididymides, ovaries.

Postmortem examinations (offspring):

On Lactation Day 5-7 external macroscopic examination of all survivors for gross abnormalities.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data
(scores) in the summary tables. Test statistics were calculated on the basis of exact values for means
and pooled variances. Individual values, means and standard deviations may have been rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter, yet
display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See 'Tables_BW_OECD421_T-1063FM.pdf' attached as background material.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See 'Tables_food_consumption_OECD421_T-1063FM.pdf' attached as background material.

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The red foci seen macroscopically in the stomach of five high dose males correlated with minimal hemorrhage in three animals; the thickening in one animal correlated with mild inflammation.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

spermatogenic staging profiles were normal for all males examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites were unaffected by treatment. Testes and epididymis weights not affected, absence of histopathological findings in testes, epididymis and ovaries.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL = highest dose tested. The findings in the stomachs of five high dose males were of mild severity and not considered to be adverse.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL = highest dose tested.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

See tables attached as background material.

Applicant's summary and conclusion

Conclusions:

In this screening study there were no treatment related effects on reproduction or developmental toxicology parameters. The no-observed-adverse- effect-levels (NOAEL) for parental toxicity regarding reprotoxic endpoints and for foetal toxicity are 1000 mg/kg bw/day. The NOAEL for general parental toxicity also was derived at 1000 mg/kg bw/day.