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

Developmental toxicity / teratogenicity

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same 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:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

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

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.

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%.

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.

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:

Once daily for 7 d/w.

Duration of test:

Males: 29 days
Females: 41-54 days

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

Examinations

Maternal examinations:

Clinical observations performed and frequency:
- Clinical signs: Daily (covering external appearance, motor activity & morbidity in each animal)
- 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, Females: Weekly for pre-pairing period and for Gestation Days 0, 4, 7, 11, 14, 17 and 20and on Lactation Days 1 and 4

Ovaries and uterine content:

Examinations included:
- Number of corpora lutea
- Number of implantations

Fetal examinations:

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.

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.

Indices:

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: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

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: foetal toxicity

Fetal abnormalities

Key result

Abnormalities:

not examined

Localisation:

other: no specific examination in this screening study; pups did not show abnormalities

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

See tables attached as background material on pup body weight and litter size.

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.