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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2022-07-13 to 2023-01-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2023
Report date:
2023

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium salts of [[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid (5-7 Na:1)
EC Number:
701-216-4
Molecular formula:
DTPMP-5Na C9H23N3Na5O15P5 DTPMP-6Na C9H22N3Na6O15P5 DTPMP-7Na C9H21N3Na7O15P5
IUPAC Name:
Sodium salts of [[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid (5-7 Na:1)
Test material form:
liquid

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
The rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for embryo-foetal development toxicity testing by regulatory agencies. Currently live animals have to be used; as acceptable models which do not use live animals currently do not exist.

TEST ANIMALS
- Source: Charles River Laboratories, 2109 Rte de Chatillion, 01400 Romans, France
- Age at study initiation: 18-20 weeks
- Weight at study initiation: 3193- 4309 g
- Fasting period before study: No
- Housing: Animals were housed singly in composite plastic and metal cages that were colour coded to indicate the study, group, animal number, and sex. Cages were arranged in order of group.
- Diet (e.g. ad libitum): Pelleted complete diet-3409 KLIBA, 200 g/animal/day
- Water (e.g. ad libitum): Softened and filtered municipal drinking water, ad libitum (except during procedures)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): ≥ 35%
- Air changes (per hr): 10 or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light: 12 hours dark

IN-LIFE DATES: From: 24-27/07/2022 To: 19/08/2022

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dose formulations were prepared on a weekly basis and divided into aliquots to be dispensed on each dosing occasion. These were prepared at appropriate concentrations to meet dose level requirements according to the standard operating procedures of the test facility. The formulation was stirred by magnetic stirrer until completion of treatment (except during delivery to the animal facility).

The method for administering the substance was oral gavage.
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: 0, 10, 30, 90 mg/mL
- Amount of vehicle (if gavage): 4mL/kg/day
- Lot/batch no. (if required): Not specified
- Purity: Not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by a total organic carbon analyser using a validated analytical procedure.
Averaged results from homogeneity analysis served as concentration verification
Details on mating procedure:
Females were mated by the supplier and delivered on GD0
Duration of treatment / exposure:
GD 6-28
Frequency of treatment:
Daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
Mid dose group
Dose / conc.:
360 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
22F
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected following dose range finding toxicity study following oral administration in white New Zealand rabbits. During this study 6 females/group were treated at dose levels of 300, 400, and 500 mg/kg bw/day from GD 6-27. Dose levels equal to or above 400 mg/kg bw/day were considered above the maximum tolerated dose as both dose levels, 400 and 500 mg/kg bw/day were associated with premature death of 2 out of 6 females after incurring marked body weight loss. Amongst the surviving animals, there was a slight to marked dose-related mean body weight loss and a dose-related progressive marked reduction in mean food consumption throughout the dosing period leading to mean food intake <50 g/day towards the end of the gestation period. There was no test item related maternal toxicity at 300 mg/kg bw/day and no clear test item related embryo-foetal effects in any group, based on this the high dose was chosen to be 360 mg/kg bw/day, and the low and mid doses were chosen to be 40 and 120 mg/kg bw/day respectively.
- Rationale for animal assignment: Random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before and at least once after dosing, and at least once daily on non-dosing days


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A full clinical examination was performed at least on each weighing day - animals removed from cage

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 3, 6, 9, 12, 15, 18, 21, 27, and 29

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Ovaries, uterus

OTHER: Mortality
Animals checked for mortality at least twice a day; at the start and end of each working day
Ovaries and uterine content:
-Ovaries and uterus were removed and examined including examination of the placentae.
-The gravid uterus weight was recorded
-The number and distribution of intrauterine implantations was recorded
-These implantations were classified as: Live foetuses, dead foetuses, early resorptions, late resorptions, and empty implantation sites and recorded
Blood sampling:
-Not examined
Fetal examinations:
-Foetal weights and sex were recorded
-Each foetus was examined for external defects
-All live foetuses were examined viscerally
-Half of the foetuses heads were removed and fixed for subsequent examination by serial sectioning.
-The eviscerated foetal carcasses were fixed and processed for skeletal examinations.
-Dead foetuses were examined externally and preserved in Harrison's fixative but not examined any further.
Statistics:
Means, standard deviations percentages, numbers and /or incidences were reported as appropriate by data set.
-All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using 2 sided tests and were reported at the 1% and 5% levels.
The pairwise comparisons were:
Group 2 vs Group 1
Group 3 vs Group 1
Group 4 vs Group 1

Analyses were further performed using parametric and non-parametric tests.
Levene's test was used to assess the homogeneity of group variances. Groups were then compared using an overall one way ANOVA F-test if Levene's test was not significant or the Kruskal-Wallis test if it was significant.
If the overall F-test or Kruskal-Wallis test was found significant, then pairwise comparisons were conducted using Dunnett's or Dunn's tests respectively.
A Fisher's exact test was used to conduct pairwise group comparisons of interest.
Indices:
Gravid uterus adjusted body weight: Terminal body weight- Gravid uterus weight
Pre-implantation loss (%): ((Number or corpora lutea- Number of implants)/ Number of corpora lutea)*100
Post-implantation loss (%): ((Number or implants- Number of live foetuses)/ Number of implants)*100
Sex-ratio (% live males): (Number of live male foetuses/ Total number of live foetuses)*100
Litter % of foetuses with abnormalities: (Number of foetuses in litter with a given finding/ Number of foetuses in litter examined)*100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Associated with reduced food consumption there was a dose-related higher incidence of females with reduced faecal output and/or soft faeces in all treated groups compared with the control group.
Other effects non-treatment related that were observed include: salivation, fur loss/ thin cover/ ungroomed/staining, skin lesions/discoloured/scab/staining, swollen vulva, protruding eyeball, discharge, broken teeth and/or absent urine. These occurred sporadically across the groups and were considered incidental or related to pregnancy rather than related to the test item.
Dermal irritation (if dermal study):
effects observed, non-treatment-related
Description (incidence and severity):
It was noted that skin lesions, discolouration, scabs and staining occurred over the course of the experiment this, however, was considered to be incidental or related to pregnancy rather than the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were 5 animals which were preterminally euthanised due largely to low food consumption and abortion.
This included one control group female that was aborted on day 28 following low food consumption, body weight loss from GD12 and red discharge with aborted tissue being present in the cage on GD 28.
One female in the 40 mg/kg bw/day dosing group that had chronic anorexia with low body gain or body weight loss from GD 0 and red discharge with aborted tissue being present in the cage on GD 24.
3 females in the 360 mg/kg bw/day group also had low food consumption from GD 12,9, and 9 respectively, with low body gain or body weight loss from GD 6, 0, and 0 respectively with red discharge and aborted tissue being present in the cage from GD 23, 25, and 25.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a test item related lower mean body weight gain or body weight loss in all treated groups during the overall dosing period compared to the control over the whole dosing period. This can be seen in the mean body weight gain from GD 6 to 29, with the control group having a mean gain of 451.1 g, the 40 mg/kg bw/day group having a mean gain of 333.2 g, the 120 mg/kg bw/day group having a mean gain of 354.4 g and the 360 mg/kg bw/day group having a mean gain of 294.2g. This can also be seen in the mean terminal body weight, where the 120 mg/kg bw/day group have a reduction of 5.7% mean terminal body weight compared to the control, and the 360 mg/kg bw/day group have a reduction of 8.5%.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a marked reduction in mean food consumption across the dosing groups from GD21, GD12, and GD6 in the 40, 120, and 360 mg/kg bw/day groups respectively. Between GD21 and GD29 there was a reduction compared to the control group of 6.5%, 15%, and 17.6% in the 40, 120, and 360 mg/kg bw/day groups.
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:
effects observed, treatment-related
Description (incidence and severity):
There was a reduced gravid uterus weight of the high dose group compared to the control, of 475g compared to 568g respectively. There was no test-item related effect on the mid and low dose groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
2 females in the high dose group were observed to have pale liver, and a cyst on the oviduct was observed in one female from each treated group, however, these findings are incidental
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
5 abortions were seen over a range of dosage groups during the study. These abortions were largely associated with low food consumption and low body weight gain, or body weight loss. These abortions were seen in the control group, the dose group, and the high dose group with 1 abortion, 1 abortion, and 3 abortions respectively. In the control group this abortion followed low followed low food consumption and body weight loss from GD12, this is considered incidental. In the low dose group the abortion occurred following chronic anorexia from the acclimatisation phase, this is thought to be related to poor acclimatisation rather than test item related. The three abortions in the high test group were considered secondary to test item related maternal toxicity, at necropsy one animal was observed having pale kidneys and liver.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Incidentally higher mean number of corpora lutea were observed in the control group compared with the historical control data, meaning that the average numbers of pre-implantation was lower across the treated groups, compared to the concurrent control. The post-implantation loss was marginally higher in the control group (11.6%) compared to the dosed groups (8.3%, 8.1%, and 5.8% - low, mid, and high respectively) but remained close to the historical control data (up to 11.5%), meaning that this finding could be considered incidental.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
1 female in the mid dose group had total litter resorption, this result was not seen in any other dose group or animals, and therefore was considered incidental.
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not examined

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Maternal toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a statistically significant lower mean foetal weight in the high dose (35.3 g) compared to the concurrent control (38.9 g) and the historical control data range (37.6-42.3 g), this is considered related to maternal toxicity, no test item related effect was observed in the mid and low dose level groups.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not specified
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group: no effects seen
In the low dose group: fused nasals.
In the mid dose group: malrotated hindlimbs, absent tail, malformed vertebral column (small, misaligned and/or misshapen thoracic centra, absent lumbar vertebrae, fused lumbar arches, absent sacral and caudal vertebrae).
In the high dose group: Malformed thoracic vertebrae (small and misaligned centrum, absent arch, branched rib; scoliosis), fused frontals, absent eye bulges, cleft face, anencephaly, malformed skull (misshapen or absent bones), short snout and malformed skull.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group: fused ribs
In the low dose group: fused nasals
In the mid dose group: malrotated hindlimbs, absent tail, malformed vertebral column
In the high dose: Malformed thoracic vertebrae, fused frontals, anencephaly, malformed skull was seen in two animals with one having misshapen or absent bones, and the other having short and misshapen nasals, full frontals and short maxilla
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group: no effects were observed
In the low dose group: malpositioned intestine, retroesophageal subclavian - in two animals, dilated aortic arch, ventricular septal defect, atretic pulmonary trunk, malformed thoracic vertebrae, and fused sternbrae.
In the mid dose group: omphalocele, retroesophageal subclavian artery
In the high dose group: anencephaly, narrowed aortic arch 3 chambered heart, dilated pulmonary trunk, retroesophageal subclavian artery

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse findings on development parameters

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a prenatal developmental toxicity study conducted according to OECD Test Guideline 414 and in compliance with GLP, daily oral gavage administration of the test item DTPMP (5 -7 Na) salt (EC: 701-216-4 ) at doses of 40, 120, and 360 mg/kg bw/day in the pregnant female New Zealand White rabbit from GD 6 -28 were associated with dose related lower mean body weight gain, and reduced mean food consumption in the 120 and 360 mg/kg bw/day groups, considered adverse at 360 mg/kg bw/day.
A no observed adverse effect level (NOAEL) for maternal toxicity was therefore set at 120 mg/kg bw/day.
There was no evidence of embryo-foetal toxicity and no test-item related external, visceral or skeletal morphological changes in any group.
Despite the lower mean pup body weight noted at 360 mg/kg bw/day probably related to the maternal toxicity, the no observed adverse effect level (NOAEL) for prenatal development was however set at 360 mg/kg bw/day.