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Key value for chemical safety assessment

Effects on fertility

Description of key information

In line with ECHA Final Decision number CCH-D-2114495769-22-01/F an extended one-generation reproductive toxicity study in rats, according to OECD Test Guideline 443, is ongoing for the Category member DTPMP (5-7Na), which will be read across to DTPMP (1-3Na). The deadline for submitting the requested information is 22 May 2023.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In line with ECHA Final Decision number CCH-D-2114495769-22-01/F an extended one-generation reproductive toxicity study in rats, according to OECD Test Guideline 443, is ongoing for the Category member DTPMP (5-7Na), which will be read across to DTPMP (1-3Na). The deadline for submitting the requested information is 22 May 2023.


 


The reproductive toxicity study with DTPMP-H in the rat (BioDynamics Inc., 1979, reliability score 2) was completed prior to the establishment of GLP and OECD guidelines. Many endpoints considered relevant to an assessment of reproductive toxicity thus are missing from the study (e.g., oestrous cyclicity and sperm parameters, pubertal assessment, anogenital distance, thyroid hormone levels). Nevertheless, the study includes a large number of females per parental generation and the production of two generations of rats. Therefore, the study provides data relevant to an assessment of developmental and reproductive toxicity. It should also be noted that, although titled as a one-generation study, the F1 animals actually were mated to produce two litters (F2a/F2b); thus, the study is more appropriately considered to be a two-generation reproductive study.


The test substance was administered in the diet to Long-Evans rats at dietary concentrations of 0, 300, 1000, and 3000 ppm beginning at the initiation of mating through the production of two generations (F1 and F2a/F2b). Test substance administration to the P0 generation females (20/group) was initiated at the onset of gestation and continued throughout the ensuing gestation and lactation periods of F1. Then, F1 rats (n=10 males and 20 females/ group) were first mated approximately 80 days after weaning, with a 14-day rest period after weaning of the first litter (F2a) before mating for production of the second litter (F2b). Rats of the F2a litters were sacrificed at weaning (postnatal day [PND] 21). Of relevance to the reproductive system, the testes and ovaries of F1 parental animals were weighed, and the following organs were preserved and evaluated histopathologically in 5 F1 adults/sex in the control and high dose groups: ovaries, testes, uterus and prostate.


No treatment-related effects were observed on parental (P0 and F1) survival, clinical signs, or food consumption. No treatment-related gross findings were reported. Both absolute and relative organ weights were comparable across the groups and no treatment-related histopathologic changes were observed in F1 animals. Although F1 female adult body weights were slightly, but not statistically, lower during lactation of the F2b litters, body weight change was generally comparable across groups and similar differences were not observed during the F2a lactation period or for P0 animals during lactation of the F1 pups. Thus, it is unlikely that this finding was related to treatment.


Most reproductive and litter parameters (mating and fertility indices, gestation length, litter size, pup sex ratio) were comparable across the groups with some minor exceptions, as detailed below.


For the F1 litter, at 3000 ppm, the mean number of live pups was slightly, but not statistically significantly, lower compared to control; the live birth index was significantly reduced at this exposure level as a result. However, differences across groups in F1 pup survival showed no clear relationship with dose and similar findings regarding live litter size were not observed for the F2a or F2b litters.


F1 pup body weights at birth were non-statistically significantly lower than control at 3000 ppm; F2a pup weights were lower at 1000 and 3000 ppm (statistically significant at 3000 ppm only). However, in both cases, pup weights during weaning were comparable across groups and a similar finding was not seen for the F2b pups.


F1 adult animals at 3000 ppm exhibited a slightly lower rate of pregnancy compared to controls for the generation of the F2a litters; however, the difference was not statistically significant, nor seen for the P0 animals or repeated with generation of the F2b litters. Additionally, F1 gestation length was reduced at 1000 ppm only for generation of the F2a litters, but not for generation of the F2b litters. Because the change in pregnancy rate was not statistically significant, and due to the lack of consistency across generations and litters, these findings are thus unlikely to have been treatment related.


In summary, the reproductive study of DTPMP-H failed to demonstrate any consistent changes indicative of a treatment-related effect. Thus, this study is supportive of there being no adverse effect of DTPMP treatment on development or reproduction at doses that did not cause toxicity in parental animals. Therefore, the reproductive and systemic NOAELs were concluded to be at least 3000 ppm, equivalent to 294 mg/kg bw/day for males and 312 mg/kg bw/day for females.


For more discussion on the findings in this study please see the Expert Review of Developmental and Reproductive Toxicity Data for ATMP, DTPMP & EDTMP (DeSesso, 2021) attached to IUCLID Section 13.


 

Effects on developmental toxicity

Description of key information

There are no prenatal developmental toxicity data for DTPMP (1-3Na), therefore data are read-across from DTPMP (5-7Na).


 


In the prenatal developmental toxicity study with DTPMP (5-7Na) (Monsanto, 1982, reliability score 2) 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 treatment-related developmental effects at the highest dose level tested. Although completed prior to the establishment of OECD guidelines, this study generally meets current guideline recommendations with the exception that the highest dose tested exceeded the limit dose of 1000 mg/kg/day. Additionally, both the in-life portion of the study (done at Monsanto) as well as the foetal examinations (done at WIL Research Laboratories) were conducted according to GLP.


 


 


A key developmental toxicity study was performed on New Zealand White Rabbits in 2023. This study was conducted according to OECD test guideline 414 and was in compliance with GLP (Charles River Laboratories, 2023, reliability 1). Female rabbits were treated daily via oral gavage with 0, 40, 120, or 369 mg/kg bw/day of DTPMP (5-7 Na). The NOAEL was calculated to be 120 mg/kg bw/day due to decreased food consumption and body weight. No adverse events were observed in the foetuses, so it was concluded that the foetal NOAEL would be greater than 360 mg/kg bw/day. 

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
GLP compliance:
yes
Limit test:
no
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
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
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
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
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
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
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
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
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
Key result
Developmental effects observed:
no
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.
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
Remarks:
The restrictions were that there was no analytical confirmation of dosing solutions, and there was no record of gravid uterine weight or the number of corpora lutea.
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
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
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 equivalent 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
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
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.
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
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 (ninth day of treatment) and persisted through to gestation day 17. This finding was not observed in the other treated groups or the controls.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
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):
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:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
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:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
not specified
Description (incidence and severity):
There were no statistically significant treatment related effects on pre and post implantation loss observed for any of the treatment groups.
Total litter losses by resorption:
not examined
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:
not examined
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:
not examined
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
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
other: maternal toxicity
Abnormalities:
effects observed, treatment-related
Localisation:
other: body weight gain
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):
males/litter: 5.6/5.8/5.9/4.8. Females/litter: 5.9/6.5/5.4/6.4
There was no effect on sex ratios.
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:
no effects observed
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 occurrence 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
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Abnormalities:
no effects observed
Developmental effects observed:
no
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-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.
Executive summary:

In a good quality prenatal developmental toxicity screening study (reliability score 2) conducted using a protocol similar to OECD 414, and to GLP, mated female Sprague-Dawley rats (25/dose) were dosed by gavage at doses of 500, 1000, 2000 mg/kg bw/day (neutral sodium salt; expressed as active acid). They were dosed on gestation days 6 to 19. Control animals were given 0.9% sodium chloride solution. On day 20 all surviving animals were sacrificed. There were no deaths prior to scheduled sacrifice. The highest dose produced marginal toxic effects in the dams, as evidenced by lower mean body weight gain between gestational days 6 and 20, and soft stools in some animals. No treatment-related lesions were detected at gross necropsy of the dams of any treatment group.

There were no statistically significant treatment-related effects on postimplantation loss or foetal weight at any dose. Vertebral anomalies were observed in single foetuses in two litters of the 2000 mg/kg bw/day group and one of the 1000 mg/kg bw/day group. The incidence was not statistically significant, but the rare spontaneous occurrence of this type of malformation and the pattern of incidence was suggestive of a treatment-related effect. 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.

Overall, the NOAEL for maternal toxicity was 1000 mg/kg bw/day, and for developmental toxicity was 2000 mg/kg bw/day (active acid).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There are no prenatal developmental toxicity data for DTPMP (1-3Na), therefore data are read-across from other DTPMP Category members. See attachment to Section 13 for justification of read-across.


 


In the prenatal developmental toxicity study with DTPMP (5-7Na) (Monsanto, 1982, reliability score 2) mated female Sprague-Dawley rats (25/dose) were given daily oral gavage doses of 500, 1000, 2000 mg/kg bw/day DTPMP (5-7Na) in 0.9% sodium chloride (expressed as active acid) during gestation days 6 to 19. Control animals were given 0.9% sodium chloride solution. On day 20 all surviving animals were sacrificed and foetuses were examined. Although completed prior to the establishment of OECD guidelines, this study generally meets current guideline recommendations with the exception that the highest dose tested exceeded the limit dose of 1000 mg/kg bw/day. Additionally, both the in-life portion of the study (conducted at Monsanto) as well as the foetal examinations (conducted at WIL Research Laboratories) were conducted according to GLP.


There were no deaths prior to scheduled sacrifice. Findings consistent with maternal toxicity were noted at the highest dose tested (2000 mg/kg bw/day), including soft stools beginning GD 14, and a reduction in maternal body weight gain of approximately 32%; however, terminal body weights were only marginally different from control (3%-4%). No treatment-related lesions were detected at gross necropsy of the dams of any treatment group. All groups generally showed low pregnancy rates, but since mating occurred prior to dosing, this finding is not related to treatment. No significant treatment-related effects were observed on pregnancy or litter parameters, although foetal weights at the high dose were approximately 7% lower than control. No external malformations were observed at the foetal examinations. A single foetus with hydrocephalus and a single foetus with gastroschisis were reported at the middle dose of 1000 mg/kg bw/day; gastroschisis (in which the intestines protrude from the abdominal cavity) is a finding typically observed at external examination, so its reporting as a visceral finding only is unusual. Importantly, no visceral findings were observed at 2000 mg/kg bw/day. Few skeletal anomalies were observed in the study; these findings occurred at single instances and/or without dose-response, and thus, their relation to treatment is unlikely. Among the skeletal observations are vertebral anomalies in one and two foetuses at 1000 and 2000 mg/kg bw/day, respectively, the incidence of which was not statistically significant and the effect was only observed in the presence of maternal toxicity. These findings involve minor reductions in ossification, extra ossification centres and bony attachments between bones that are likely transient. Few foetal variations were reported and those of the skeletal system primarily related to the degree of bone ossification (data not shown). None of the variations were observed with a dose-response with the exception of unossified sternebrae #5 and/or #6, which was increased in foetal incidence at the high dose. In summary, these data show that no treatment-related developmental toxicity occurred at DTPMP doses above the limit dose of 1000 mg/kg bw/day (2000 mg/kg bw/day). The NOAEL for maternal toxicity was concluded to be 1000 mg/kg bw/day based on the lower body weight gain, while the NOAEL for developmental toxicity was concluded to be 2000 mg/kg bw/day (active acid) based on no adverse treatment-related developmental effects.


For more discussion on the findings in this study please see the Expert Review of Developmental and Reproductive Toxicity Data for ATMP, DTPMP & EDTMP (DeSesso, 2021) attached to IUCLID Section 13.


 


In the developmental toxicity study with DTPMP (5-7Na) (Charles River Laboratories, 2023) New Zealand White Rabbits (22/dose) were given daily oral gavage doses of 0, 40, 120, or 360 mg/kg bw/day DTPMP (5-7Na) in water during gestation days 6-28. The females were then sacrificed and foetuses were examined. This study was conducted according to OECD Test Guideline 414 and was completed in compliance with GLP.


 


There were 5 deaths prior to the scheduled sacrifice. These animals were euthanised due to low food consumption and abortion. These animals included one in the control group, one in the low dose group and three in the high dose group. Findings that have been related with maternal toxicity were observed including reduced food consumption, reduced faecal output or soft faeces were observed in a dose related manner compared to the control group. There was also reduced body weight gains that was attributed to the test item compared to the control group and can be seen in the mean terminal weights and in the weight gain patterns observed between days 6-29. 


 


Reduced gravid uterus and reduced mean foetal body weight were associated with the higher dose group; however, no other statistically significant observations were made with regards to the foetuses. Whilst there were external, skeletal, and visceral malformations observed these incidences were not seen in a dose dependant manner and were attributed to a single litter, this strain of rabbit is associated with these malformations. 

Justification for classification or non-classification

Based on the available data, no classification for reproductive and developmental toxicity is required for DTPMP (1-3Na) according to Regulation (EC) No 1272/2008.

Additional information