[nitrilotris(methylene)]trisphosphonic acid, ammonium salt

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data. Dates of treatment were 27.12.1978 to 19.01.1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The study is a read across from ATMP (CAS 6419-19-8).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Guideline:

other: FDA "Guidelines for reproductive studies for evaluation of drugs for human use", segment II (teratological study)

Deviations:

not specified

Remarks:

Treatment on GD 6 - 15; no record of gravid uterine weight; number corpora lutea not recorded; no analytical confirmation of exposure levels.

Principles of method if other than guideline:

Study was used to assess the teratogenic and/or embryotoxic potential of the test substance.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

1

Reference substance name:

[nitrilotris(methylene)]trisphosphonic acid, sodium salt

EC Number:

243-900-0

EC Name:

[nitrilotris(methylene)]trisphosphonic acid, sodium salt

Cas Number:

20592-85-2

Molecular formula:

General formula C3H12NO9P3.xNa where x=3-5 ATMP-3Na C3H9NNa3O9P3 ATMP-4Na C3H8NNa4O9P3 ATMP-5Na C3H7NNa5O9P3

IUPAC Name:

Sodium salt of [nitrilotris(methylene)]trisphosphonic acid (3-5Na:1)

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

72 d at mating
Weight: approx. 240 - 250 Number: 24 / dose Supplier: Charles River, Wilmington, Mass. Individually housed, food (Purina Certified Rodent Chow 5002) and water available ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

ADMINISTRATION / EXPOSURE GD 6-15 Test substance mixed with water, administered at 10 mg/kg/d. Volume adjusted based on most recent body weight data. Prepared daily.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Overnight
- Further matings after two unsuccessful attempts: No data
- Verification of same strain and source of both sexes: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.

Duration of treatment / exposure:

GD 6 - 15

Frequency of treatment:

daily

Duration of test:

16 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

measured as the active acid

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

measured as the active acid

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

measured as the active acid

No. of animals per sex per dose:

24 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: 21 d

Examinations

Maternal examinations:

Clinical observations performed and frequency: - gross signs, twice daily - detailed physical examination of GD 0, 6, 10, 15, 20 and 21 (pre-necropsy) Parent: - bwt: GD 0, 6 -15, 21
- Organs examined at necropsy Dams sacrificed on GD21 by lethal exposure to diethyl ether.
Parent: - complete post-mortem examination - uterus: live / dead foetuses, late / early resorptions, implantation sites - ovaries: corporea lutea per ovary

Fetal examinations:

Fetal: - crown-rump distance - sex (anogenital distance) - external malformations - approx. 50% of foetuses subject to gross dissection and visceral examination followed by processing / staining (Alizarin red) for skeletal abnormalities / variations - remainder subject to Wilson serial sectioning for neural / visceral defects (10X or 20X magnification) after fixing in Bouin's solution

Statistics:

Chi squared or F-test and Student's T-test. T-tests modified using Cochran's approximation when variances differed significantly. Live foetuses, resorptions, implantations and corpora lutea compared using one-tailed T-test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs in any dose groups were found.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 100 mg/kg bw/day dose group was concluded to be in moribund condition and was sacrificed on GD6 (first day of treatment).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant differences were found in maternal body weight gain between groups. The body weight gain GD6-15: 50/49/50/44 - 12% (non-significant) reduction in body weight gain at 1000 mg/kg bw/d on GD6-15. Individual body weight gain for dam 822 (high dose) on GD6-15 = 22 g; mean gain for controls = 50 g; mean gain for high dose group = 44 g. Body weight gain for this dam on GD 0-6 (preceding treatment) and on GD 15-21 (post-treatment) was similar or greater than mean bdoy weight gain for control and high dose group.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed in any of the dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

A statistical significant 7.5 % increase in implantation efficiency at 100 mg/kg bw/day was observed. However, this was considered unrelated to the test substance by authors as the implantation (14.5/14.7/14.0/13.7) and implantation efficiency (84.3%/91.8%/84.0%/81.2%) was comparable.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Mean number of resorptions were 0.2/0.8/0.5/0.8 which were all within historical control range data, There was an increase although not statistical significant increase in dams with 2 or more resorptions in treated groups (0%/22.7%/8.3%/20.8%), However, this was also within historical control range and therefore, considered to not be test substance treatment-related effects.

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses were observed in any of the dose groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The pregnancy rate was comparable between all dose groups (100% in control, mid and high dose groups, 95.6% at 100 mg/kg bw/d).

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of corpora lutea were 17.2/16.0/16.7/16.8. The 100 mg/kg bw/day dose group had a 7% decrease in corpora lutea. However, this was considered by the authors to be unrelated to the treatment

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

act. ingr.

Remarks:

active acid

Basis for effect level:

other: Effects on body weigh gain.

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not specified

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex: males/litter 6.9/7.0/6.6/6.0; females 7.4/6.9/7.0/6.9 (no significant effect). Sex ratio (m:f): 92.4%/102.0%/94.6%/87.3% (no significant effect)

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The body weight for males were 5.54/5.43/5.71/5.49 and for females 5.25/5.17/5.34/5.16 which did not indicate any significant effect.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 mg/kg bw/day dose group, one female had 6 foetuses (from a total litter of 16) with a syndrome of defects that included: flexed forepaws, shortened and thickened torso, abdominal distension and exaggerated forward flexure of the head (see maternal body weight, above). No other animals, including the other animals in the 1000 mg/kg bw/day dose group, had any external malformations and thereby, this effect were considered to be incidental and not related to treatment of the test substance.

Description (incidence and severity):

Total of foetuses examined: 177/158/169/159 - per foetus: 4.0%/1.9%/3.0%/1.3% (no significant effect) - per litter: 20.8%/13.6%/20.8%/8.7% (no significant effect). In the control dose group, angulated ribs, cervical rib and wavy rib were observed. In the 100 mg/kg bw/day dose group, angulated rib, cervical rib as well as angulated and wavy rib were observed. In the 500 mg/kg bw/day dose group. cervical rib, angulated and wavy rib as well as 7 lumbar vertebra were observed. In the 1000 mg/kg bw/day dose group, 5 lumbar vertebra and fused sternebrae were observed. There was no significant variation in the ossification: fetuses 80.2%/83.5%/79.3%/84.3%; litters 95.8%/100.0%/100.0%/100.0%.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Total foetuses examined: 162/147/156/150 - per fetus: 4.3%/8.2%/4.5%/4.0% (no significant effect) - per litter: 16.7%/40.9%/29.2%/20.8% (no significant effect). In the control dose group, distended renal pelvis, renal pelvis, ureter and bladder malformations were observed. In the 100 mg/kg bw/day dose group, a similar incidence in the control dose group as well as a fold in retina. In the 500 mg/kg bw/day dose group, a similar incidence as in the control dose group as well as ectopic kidney. In the 1000 mg/kg bw/day dose group, a similar incidence as in the control dose group as well as fold in retina, anophthalmia, malrotation of heart occurring in two foetuses from the same litter which had an altered maternal body weight and observed external malformations.
The incidence of visceral malformations per fetus were: 0.6%/1.3%/0.6%/1.9% (no significant effect) - per litter: 4.2%/9.1%/4.2%/12.% (no significant effect). In the control dose group, distended ureter was observed. In the 100 mg/kg bw/day, a distended ureter +/- renal pelvis occurred. In the 500 mg/kg bw/day dose group, a similar incidence occurred as in the control dose group. In the 1000 mg/kg bw/day, a similar incidence occurred as in the control dose group as well as malpositioned testis were observed.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Crown-rump length: males 4.2/4.2/4.3 (P<0.01)/4.2; females 4.1/4.1/4.2 (P<0.01)/4.1. There was an increase at 500 mg/kg bw/day, however, this was not considered be of biological insignificance by authors.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No teratogenic effects observed.

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No fetotoxic effects observed.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a well-documented pre-GLP teratology study (FDA segment II teratological study; reliability score 2) ATMP-H (aqueous solution containing 22.4% w/w active acid) was not embryotoxic or teratogenic when administered to rats at 100 or 500 mg active acid/kg bw/day (measured as the active acid) by gavage on GD6-15. At 1000 mg/kg bw/d, six fetuses from a single litter showed common multiple malformations in presence of a 50% decrease in individual maternal body weight gain (possibly indicative of concurrent maternal toxicity); all other high dose fetuses were normal. The clear absence of any comparable effect in other high dose litters and lack of dose-response indicates that 1000 mg active acid/kg bwt/d was the no-effect level for embryotoxicity and fetotoxicity. The maternal NOAEL was 500 mg active acid/kg bw/day.