Sodium salts of [[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid (5-7 Na:1)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.05.1980 to 09.06.1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

No record of gravid uterine weight, number of corpora lutea not recorded, no analytical confirmation of dosing solutions.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

octadecasodium ({[2-({2-[bis(hydrogen phosphonatomethyl)amino]ethyl}(hydrogen phosphonatomethyl)amino)ethyl](hydrogen phosphonatomethyl)amino}methyl)phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonatomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate

EC Number:

701-216-4

Molecular formula:

DTPMP-5Na C9H23N3Na5O15P5 DTPMP-6Na C9H22N3Na6O15P5 DTPMP-7Na C9H21N3Na7O15P5

IUPAC Name:

octadecasodium ({[2-({2-[bis(hydrogen phosphonatomethyl)amino]ethyl}(hydrogen phosphonatomethyl)amino)ethyl](hydrogen phosphonatomethyl)amino}methyl)phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonatomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate {[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)({2-[(hydrogen phosphonatomethyl)(phosphonomethyl)amino]ethyl})amino]ethyl})amino]methyl}phosphonate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: No data
- Weight at study initiation: 180-200 g
- Fasting period before study: No data
- Housing: Individually in suspended stainless steel mesh cages.
- Diet (e.g. ad libitum): Purina certified rodent chow #5002, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: None, animals were received mated and allocated to cages.


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ±2
- Humidity (%): 40-60
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 26.05.1980 To: 09.06.1980

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqueous solution

Details on exposure:

Animals were assigned to the control or treatment groups by a randomisation procedure giving eqivalent body weight distribution.
Animals were dosed by gavage on gestation days 6-19 for all groups contained in 8 ml dose volume/ kg body weight/day. The body weights that were used for dose calculation were the most recent body weights measured on gestation days 6, 8, 10, and 13.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

No data. Animals were received on gestational day 1, having already been mated.

Duration of treatment / exposure:

GD 6 - 19

Frequency of treatment:

daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

other: 0.9% (w/v) aqueous NaCl

Details on study design:

- Dose selection rationale: No data
-On gestation day 20 all surviving animals were sacrificed via exposure to chloroform.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for visible toxic effects.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: Gestational days 3, 6, 8, 10, 13, 15, 17 and 20.


FOOD CONSUMPTION: No


WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Gross examination of all animals that included examination of external surfaces and thoracic and abdominal cavities.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

Comparison of body weights between treatment and control groups was performed using Dunnett's test. Counted data (corpora lutea, implants, resorptions, live and dead foetuses) and data expressed as percentage were analysed, when appropriate with the Mann-Whitney U test. Response data (pregnancy rates, number of litters with post-implantation loss, and foetuses or litters with abnormalities and variants) were analysed, when appropriate, with Fisher's exact test and the chi-square test.
Standard errors of the mean were reported as more appropriate measures of variance for counted data and such data expressed as percentages. Also analysis of categorical data was performed on actual count data rather than such data expressed as a percentage.
Additional statistical tests were performed on the incidence of malformations and variants in foetuses and litters. These tests, the uncorrected chi-square test (except where low incidence frequency prevented valid analysis) and a one sided Fisher's exact test were performed.

Indices:

None

Historical control data:

No data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

9/25 dams had soft stools in the 2000 mg/kg bw/day group beginning on gestation day 14 (9th day of the treatment) and persisted through to gestation day 17. This finding was not observed in the other treated groups or the controls.

Mortality:

no mortality observed

Description (incidence):

No deaths occurred prior to the scheduled sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The only statistically significant (P<0.01) effect observed was lower body weight gain (mean value approximately 68% of the control mean) between gestation day 6 and 20 for dams in the 2000 mg/kg bw/day group (33.6/27.6/27.5/22.9). However, terminal body weights were not statistically significantly affected (mean terminal body weights with uterine contents by dose: 360.1/368.1/357.0/346.1 and mean terminal body weights without uterine contents: 265.8/264.5/260.1/257.2).

Food consumption and compound intake (if feeding study):

no effects observed

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:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

One non-pregnant female in the control group was observed to have a white mass in the bladder, hydronephrosis of the kidneys, enlarged spleen and vaginal mass. 2 females in the 0.5 kg group were observed to have ear tag infections. These were not considered to be treatment related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no significant differences between any of the treatment groups and the control group in the number of pre- or postimplantation losses.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no significant differences between any of the treatment groups and the control group in the resorptions.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no significant differences between any of the treatment groups and the control group in the resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no significant differences between any of the treatment groups and the control group in the mean number of live foetuses.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no significant differences between any of the treatment groups and the control groups in the pregnancy rate.

Other effects:

no effects observed

Description (incidence and severity):

There was no statistically significant differences in the live foetus weights observed in any of the treatment groups.
There was no effect on the mean number of corpora lutea

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

active acid

Basis for effect level:

body weight and weight gain

Maternal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

other: body weight gain

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean foetus weights were somewhat lower that control values in the 2 g/kg dose level group. Individual mean foetal weights suggested a bimodal distribution of weights with about half the litters having foetuses with 3-5 g mean weights and the other half with mean foetal weights if 5-6.4 g. The bimodal distribution may reflect a difference in the actual age of the foetuses.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no effects on sex ratios (Males/litter: 5.6/5.8/5.9/4.8. Females/litter: 5.9/6.5/5.4/6.4).

Changes in litter size and weights:

no effects observed

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):

Subcutaneous haematomas were present in controls and all treated groups, however the incidence increased at 500 mg/kg bw/day (P<0.05) and was considered unrelated to treatment by the study authors (no dose relationship was present). Oedema was observed in four foetuses in one dam in the 0.5 kg treatment group. The occurence of one female foetus that was hydrocephalic and one female with gastroschisis in the 1000 mg/kg bw/day group was considered by the study authors to be spontaneous. The hydrocephalic female also exhibited a developmental variation (underdeveloped renal papilla) that was not considered to be related to treatment. This was considered to be iatrogenic and not treatment related.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal examination revealed single incidences of dwarfism (one female foetus of 500 mg/kg bw/day group) and fused sternebrae (one female foetus of the 2000 mg/kg bw/day group), which were considered spontaneous. Two foetuses from different litters in the 2000 mg/kg bw/day group and one foetus of the 1000 mg/kg bw/day group had vertebral anomalies (missing, reduced or fused vertebral arches). Although the incidence of these anomalies was not statistically significant compared with the control group, the rare spontaneous occurrence of such anomalies and the pattern of incidence indicated that they might have been treatment-related. Various skeletal variations occurred, but none showed a clear dose-response and so were considered spontaneous. Review of these results for the REACH assessment concluded that the effects should not be considered adverse as they were not statistically significant and were only observed in the presence of maternal toxicity.

Visceral malformations:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No adverse developmental effects observed.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In the prenatal developmental toxicity study with DTPMP (5-7Na), conducted according to a protocol similar to OECD Test Guideline 414 and in compliance with GLP, clear maternal toxicity (approx. 30% decrease in body weight gain, soft stools) was noted in pregnant Sprague-Dawley rats given 2000 mg/kg bw/day DTPMP (5-7Na) (expressed as active acid) on gestation days 6-19, therefore the NOAEL for maternal toxicity was concluded to be 1000 mg/kg bw/day. The NOAEL for developmental toxicity was concluded to be 2000 mg/kg bw/day (active acid) based on no adverse developmental effects.