Tetrasodium (1-hydroxyethylidene)bisphosphonate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.03.1985 to 17.04.1985 (inclusive of limit, range-finder and LD50 tests)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

no post-dose fast

Principles of method if other than guideline:

Bio/dynamics protocol based on US guidelines. Broadly compatible with OECD guideline.

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium (1-hydroxyethylidene)bisphosphonate

EC Number:

223-267-7

EC Name:

Tetrasodium (1-hydroxyethylidene)bisphosphonate

Cas Number:

3794-83-0

Molecular formula:

C2H4Na4O7P2

IUPAC Name:

tetrasodium (1-hydroxyethane-1,1-diyl)bis(phosphonate)

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 9-12 weeks
- Weight at study initiation: 220-329 g
- Fasting period before study: 18 hours overnight prior to dosing
- Housing: Six per cage (suspended, stainless steel with wire mesh bottoms
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 9 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 67-76
- Humidity (%): 30-70
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: Limit test: From: 21.03.85 To: 22.03.85
Range-finding test: From: 27.03.85 To: 03.04.85
LD50 test: From: 03.04.85 To: 17.03.85

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 3.8 ml/kg in the limit test, 1.5 ml/kg in the range-finding test, and 3.1 ml/kg in the LD50 test.

Doses:

Limit test: 5000 mg/kg bw.
Range-finding test: 50, 100, 500, 1000 and 2000 mg/kg bw
LD50 determination: 2000, 2500, 3200 and 4000 mg/kg bw

No. of animals per sex per dose:

Limit test: 5
Range-finding test: 1
LD50 determination: 5

Control animals:

no

Details on study design:

Animals used in the range-finding test were observed for viability twice daily and deaths were recorded. The following observations were made on all other animals:
Viability check: twice daily.
Clinical signs: approximately 1, 2 and 4 hours after dosing and daily thereafter for 14 days
Body weights: pre-fasting, post-fast, just prior to dosing), and Days 7 and 14 after dosing.
Macroscopic examination: Not on range-finding test animals. A macroscopic examination was conducted on all other animals.

Statistics:

The LD50 with 95% confidence limits was calculated using the method of Miller, Lloyd C. and M.L. Tainter., Estimation of the ED50 and its error by means of Logarithmic-Probit graph paper, Proc.Soc.Exp.Bio.Med. 57: 261-264 (1944).

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 850 mg/kg bw

95% CL:

>= 2 489 - <= 3 211

Remarks on result:

other: presumed equivalent to 940 mg active salt/kg bw

Mortality:

RANGE FINDING TEST: No deaths at any dose level. 
LD50 DETERMINATION TEST/LIMIT TEST At 2000 mg/kg, 1 animal died (day 3 after dosing) 
At 2500 mg/kg, 4 animals died (1-4 hours after dosing) 
At 3200 mg/kg, 7 animals died (1 hour - day 8 after dosing) 
At 4000 mg/kg, 8 animals died (1-4 hours after dosing) 
At 5000 mg/kg, all 10 animals died (2-7 hours after dosing)

Clinical signs:

other: LD50 determination: Observations in all groups for up to four hours post-dosing, included ataxia and/or tremors, oral and nasal discharge, hypoactivity, soft stool and fecal and/or urinary staining.  

Gross pathology:

LD50 determination: Macroscopic abnormalities observed at necropsy were primarily changes to the lungs (discolouration) and gastrointestinal tract (red or black walls, or red or black fluid present, suggestive of an irritant effect).  Most of the animals in the 2500, 3200 and 4000 mg/kg dose groups had enlarged kidneys at necropsy, and one animal in the 4000 mg/kg bw group had unilateral renal pallor, dilated renal pelvis and red fluid surrounding the kidney. 

Other findings:

None reported.

Any other information on results incl. tables

Table 1 Summary of deaths in LD₅₀ determination study.

Dose	Mortality			Time to death
mg/kg bw	Males	Females	Combined
2000	1/5	0/5	1	day 3
2500	1/5	3/5	4	1 -4 h
3200	3/5	4/5	7	1 h to day 8
4000	4/5	4/5	8	1 -4 h
5000	5/5	5/5	10	2 -7 h
LD50	3100	2500	2850
95% conf limit	2365 -3635	1802 -3198	2489 -3211

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Based on the results of a well conducted acute oral study (reliability score 2), conducted using a protocol similar to the now deleted OECD 401, but not to GLP, an LD50 of 2850 mg/kg (reviewer for IUCLID 4 comment: presumed equivalent to 940 mg active salt/kg bw) was determined for Dequest 2016 in the rat.

Executive summary:

In a well conducted, non-GLP study, 5000 mg/kg bw undiluted Dequest 2016 was given to five Sprague-Dawley rats by oral gavage in a limit test. Within seven hours all of the animals had died. Therefore a range-finding study and subsequent LD₅₀ determination were performed. In the range-finding study, doses of 50, 100, 500, 1000 and 2000 mg/kg bw were administered to one male and one female Sprague-Dawley rat. Based on the findings of this study, doses of 2000, 2500, 3200 and 4000 mg/kg bw Dequest 2016 were administered to Sprague-Dawley rats (5/sex) by oral gavage. Animals were observed for 14 days following dosing, and all animal were examined macroscopically. Body weights were recorded before dosing and on days 7 and 14. One, 4, 7 and 8 animals died, respectively. Observations in all groups for up to four hours post-dosing, included ataxia and/or tremors, oral and nasal discharge, hypoactivity, soft stool and fecal and/or urinary staining. The majority of surviving animals showed some weight loss during the first week after dosing, but all animals then gained weight between days 7 and 14. Macroscopic abnormalities observed at necropsy were primarily changes to the lungs (discolouration) and gastrointestinal tract (red or black walls, or red or black fluid present, suggestive of an irritant effect).  Most of the animals in the 2500, 3200 and 4000 mg/kg dose groups had enlarged kidneys at necropsy, and one animal in the 4000 mg/kg bw group had unilateral renal pallor, dilated renal pelvis and red fluid surrounding the kidney. The LD₅₀ was calculated to be 2850 mg/kg bw (subsequently calculated to be equivalent to 940 mg active salt/kg bw).