Sodium Salts of (1-Hydroxyethylidene)bisphosphonic acid (2-3 Na:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1974-02-04 to 1974-09-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Rodent Dominant Lethal Test. A postive control substance was not included in this study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1974

Report date:

1974

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

yes

Remarks:

Postive control substance was not included in this study.

Principles of method if other than guideline:

N/A

GLP compliance:

no

Remarks:

pre-GLP

Type of assay:

rodent dominant lethal assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium Salts of (1-Hydroxyethylidene)bisphosphonic acid (2-3 Na:1)

EC Number:

701-238-4

Cas Number:

29329-71-3

Molecular formula:

HEDP-2Na C2H6Na2O7P2 HEDP-3Na C2H5Na3O7P2

IUPAC Name:

Sodium Salts of (1-Hydroxyethylidene)bisphosphonic acid (2-3 Na:1)

Test material form:

not specified

Test animals

Species:

mouse

Strain:

other: C3D2F1/J

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, Maine
- Age at study initiation: Sexually mature
- Weight at study initiation: N/A
- Assigned to test groups randomly: Yes, on the basis of body weight
- Fasting period before study: N/A
- Housing: N/A
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: One week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): N/A
- Humidity (%): N/A
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A


IN-LIFE DATES: From: N/A To: N/A

Immediately following treatment, each male was caged with two untreated females for a period of seven days, and with two fresh females the following week. This procedure was continued for a total of six weeks, thereby encompassing the entire spermatogenic cycle of the mouse, which was purported to be 35 days.
Consequently, each male was mated to twelve females over a six-week period: A total of 240 matings per dose group.
On day 13 or 14 of gestation (as measured from the mid-week of presumptive mating), the females were sacrificed. Total implants, resorptions and dead embryos were enumerated and recorded.
Various parameters such as average number of implants per female, average number of fetal deaths/female, fetal deaths per sire, and percent pregnancy per group were subjected to statistical analysis.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: N/A
- Concentration of test material in vehicle: N/A
- Amount of vehicle: 0.25 mL
- Type and concentration of dispersant aid: N/A
- Lot/batch no.: N/A
- Purity: N/A

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: N/A

DIET PREPARATION
- Rate of preparation of diet: N/A
- Mixing appropriate amounts with: N/A
- Storage temperature of food: N/A

OTHER: Each of three groups of mice received the test substance (as a single oral daily dose in 0.25 mL volumes), at levels of 20, 200 or 1000 mg/kg bw for five days. One other group received water for five days. The fifth group received no treatment.

Duration of treatment / exposure:

5 days

Frequency of treatment:

once daily

Post exposure period:

N/A

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control group, no treatment

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

Positive controls: None

Examinations

Tissues and cell types examined:

total implants, resorptions and dead embryos

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The top dose for the dominant lethal assay was based on the maximum tolerated dose determined from a preliminary study (see below under additional information on results). The intermediate dosing level was arbitrarily set at 200 mg/kg bw and the lower test group received the human-usage level of 20 mg/kg bw.

TREATMENT AND SAMPLING TIMES: N/A

DETAILS OF SLIDE PREPARATION: N/A

METHOD OF ANALYSIS: N/A

OTHER: N/A

Evaluation criteria:

N/A

Statistics:

Data from experimental groups were compared to data from control groups using the Chi-square test for significant deviations (P<= 0.05, DF=1)

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 62.5-2000 mg/kg bw
- Solubility: N/A
- Clinical signs of toxicity in test animals: 2 out of 10 mice died at the 2000 mg/kg dose level.
- Evidence of cytotoxicity in tissue analysed: N/A
- Rationale for exposure: To determine an LD50 and/or a maximum tolerated dose for mice.
- Harvest times: Animals were exposed daily for 5 consecutive days.
- High dose with and without activation: 2000 mg/kg without activation
- Other: An LD50 could not be calculated, but the maximum tolerated dose was chosen as 1000 mg/kg.


RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: Top dose was based on maximum tolerated dose.
- Statistical evaluation: N/A

Any other information on results incl. tables

Total litter losses occurred very rarely throughout the study and showed no dose response to the test substance. Only two litters approached being totally lost in all test groups. One incident occurred in the untreated controls in which two to three implants in one female were dead and another occurred in the high dose group in which only one of five implants was alive at necropsy. Both litter losses originated during the third week of mating and were attributed to a malfunction in the individual animals and therefore were not considered test-substance related. The number of deaths in females mated with males given the test substance did not differ significantly from those in concurrent controls for any of the mating periods. Mating performance of all test groups compared favorably with the controls. As in the examination of individual female data, individual poor performance of the sire which was not test related could easily be detected. For example one male in the mid test group failed to impregnate a female until the fifth week of mating. In the final two weeks of the study, three of the four females mated with this male mouse conceived but only carried an average of six implants per pregnancy. The average number of implants for the rest of the group was 8.9 for the entire study and 9.0 for all other sires combined during the same two weeks of mating. The fact that eighteen implants sired by the male mouse mentioned above produced no fetal deaths provides additional evidence supporting the decision that the performance of this male was not test related. When it came to conception rates, implants and fetal mortality, little variation occurred between test groups and controls for any of the parameters measured. The mutagenic index was measured. It was a calculation based on two independent variables, fetal deaths and total implants, used as a convenient comparison of group to group performance. No significant differences (P= 0.05. D.F=1) were found when test group mutagenic indices were compared to either the vehicle or untreated controls.

Applicant's summary and conclusion

Conclusions:

In an in vivo dominant lethal assay, conducted to a protocol similar to OECD Test Guideline 478 and pre-GLP, HEDP (2-3Na) was tested using male mice that were orally dosed once daily for five days. No test substance-related variations relative to control values in conception rates, total implant averages, foetal death averages, resorption percentage or mutagenic indices were observed. It was concluded that HEDP (2-3Na) is non-mutagenic non-mutagenic under the conditions of the study.