[[(phosphonomethyl)imino]bis[ethylenenitrilobis(methylene)]]tetrakisphosphonic acid, ammonium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no available reproductive toxicity data for DTPMP-xNH4 for the reproductive toxicity endpoint. Therefore, data were read across from the category member DTPMP-H.

The potential impact of DTPMP acid on reproductive function has been studied in a well-documented investigation in which rats were fed 300, 1000 and 3000 ppm DTPMP-H in the diet: males were treated over 1 generation (3 litters), and females over 2 generations (3 litters) (Biodynamics, 1979; Reliability 2). Treatment of the F0 generation commenced at mating and continued through gestation until weaning of the F1 pups. Treatment of the F1 generation commenced after a growth period and continued through mating, gestation and weaning over two successive litters (that is, until termination of the F2b pups). The study included an evaluation of parental mortality, growth, mating and fertility indices together with necropsy of the F1 parents. Litter parameters included gestational length, pup viability, survival and growth indices and necropsy observations at weaning. F0 females fed the high dose diet delivered litters with fewer live pups (decreased 21%) with a marginal (3%) reduction in body weight (neither effect was statistically significant). Pregnancy rate (17% reduction; not significant) and pup body weight (decreased 6.5%; statistically significant) were lower in F2a litters from dams fed 3000 ppm DTPMP, however these changes were not replicated in the F2b litters (pregnancy rate unaffected; body weight decreased 2%) suggesting they were of equivocal toxicological significance, and most probably unrelated to treatment with DTPMP. Other parental and pup parameters assess during the investigation were unaffected by treatment. A NOAEL for effects on reproductive function of 294 mg/kg bw/day (equivalent to 3000 ppm DTPMP-H in the diet) was therefore obtained for parental males and 312 mg/kg bw/day for parental females.

According to ECHA decision number CCH-D-2ii4495836-29-01/F, an extended one-generation reproductive toxicity with developmental neuro- and immunotoxicity with the registered substance DTPMP 5-7 Na-salt is currently being conducted according to OECD 443 and in compliance with GLP. The test is being conducted for a category member that is registered at Annex X. When the study result is available, the substance will be updated accordingly.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.09. 1977 to 19.06.1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

Conducted prior to adoption of OECD test guideline.

Deviations:

yes

Remarks:

No analytical confirmation of exposure, no pre-mating exposure, no assessment of oestrus cycle or sperm analyses, no evaluation of sexual milestones in pup. Males treated only as F1 and F2 generation.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Females: Blue Spruce Farms. Males: BioDynamics Inc. in-house Long Evans breeding colony.
- Age at study initiation: (P) x 15-16 wks; (F1) x 3 wks
- Weight at study initiation (means): Females approximately 240 g. Males not weighed.
- Fasting period before study: No
- Housing: Individually in elevated stainless steel cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 12.09. 1977 to 19.06.1978

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow
- Storage temperature of food: No data

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of cohabitation: nightly until signs of mating.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Individually in elevated stainless steel cages.
- Any other deviations from standard protocol: Dosing not started until Day 0 of gestation.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Test substance administration to F0 females was initiated on gestation Day 0 and continued throughout gestation and lactation, and for F1males and females through the F2a and F2b litters.

Frequency of treatment:

Daily

Details on study schedule:

- F1 parental animals not mated until one week after selection from the F1 litters.
- Selection of parents from F1 generation when pups had reached maturity.

Dose / conc.:

300 ppm (nominal)

Remarks:

in diet

Dose / conc.:

1 000 ppm (nominal)

Remarks:

in diet

Dose / conc.:

3 000 ppm (nominal)

Remarks:

in diet

No. of animals per sex per dose:

F0 females: 20.
F1 Parents: females - 20; males - 10.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random.
- F1 offspring were separated from siblings seven days after weaning of the last litter and were randomly selected to continue as future parents (f1). More offspring than needed were selected (12 males and 24 females) for the growth period to insure the required number of adults (10 males and 20 females) necessary for mating. Following pup selection, remaining offspring and F0 females were sacrificed and discarded after gross external and internal examinations.
F1 animals were raised to maturity and mated to produce the F2a litters. F2a pups were sacrificed, necropsied and discarded at weaning. All F1 females were remated after a rest period of at least 14 days to produce the F2b litters. F2b pups were sacrificed and necropsied at weaning. Following completion of the F2b sacrifice, all F1 parents were sacrificed, necropsied and selected tissues preserved.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality and gross signs of toxicity in F0 and F1: twice daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly (F0 and F1)


BODY WEIGHT: Yes
- Time schedule for examinations: Males and nonpregnant females (F1): weekly in growth and rest periods of F1 generation. Pregnant females (F0 and F1): Days 0, 6, 15 and 20 of gestation. Lactation females (F0 and F1): Days 0, 4, 14 and 21 of lactation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-Males and nonpregnant females: weekly for growth and rest periods of F1 generation.
-Test substance consumption was calculated from body weight and food consumption values: Males and nonpregnant females: weekly during growth and rest periods of F1 generation.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

Not examined as dosing in F0 dams started on Day 0 gestation, F0 males not dosed.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: No data

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED:
Gestation period: On day 19 of gestation, dams were provided with nesting material and were examined daily for signs of parturition.
Lactation period (Day 0 is day on which all pups had been delivered): General appearance of pups and presence of dead pups examined daily in all litters (F1, F2a and F2b). LIve, dead and missing pups were recorded on Days 0, 1, 4 (pre-cull and post-cull), 14 and 21 of lactation in all litters (F1, F2a and F2b). Live pups were weighed as a litter on Days 0, 4 (pre-cull) and 21 (calculated from individual weights) of lactation for all litters, and individually on Day 21 of lactation for all litters.
Litter Data and Observations: The number of each sex per litter was determined on Days 0 and 4 (pre- and post-cull) of lactation for all litters. Individual sex determination on Day 21 of lactation for all litters.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
F0 generation: All females sacrificed after weaning of F1 offspring, and necropsy performed.
F1 generation: All surviving males and females sacrificed after weaning and necropsy of the last F2b litter. Necropsy performed, and selected organs weighed and tissues preserved.
Extra F1 males and females: sacrifice at initiation of mating, necropsy performed and discarded.
Dead and moribund dams: Necropsy performed. Uterine contents examined and presence of implantation sites and/or scars recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were weighed and/or prepared for microscopic examination.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning. Necropsy performed including internal sex determination peformed, then carcassed discarded.
- F2a: necrospy included internal sex determination performed. All offspring where then discarded.
- F2b: necrospy included internal sex determination performed. Selected tissues were preserved from ten randomly chosen males and females from all groups.
F2b found dead after Day 14 of lactation: necropsy including internal sex determination performed and discarded.
Culled F1, F2a and F2b: sacrifice on Day 4 of lactation. Necropsy including internal sex determination performed and discarded.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Dead pups: sex was determined and pups checked for presence of milk in their stomach and discarded.


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 were preserved for F2b offspring.

Statistics:

Body weight, body weight gain, food consumption and litter examination data, organ weights, organ/body weight and organ/brain weight ratios were analysed. Group I and Group II (control groups) were compared to each other. If no statistically significant differences, the control groups were combined and compared against test groups. If controls differed, individual control/test comparisons conducted - continuous and discrete data (body weight, food intake, organ weight): Dunnett's test.  - gestation length: F-test and Student's T-test (Cochran's approximation). - offspring data: Chi-square. Incidence data for control Group II was compared to control Group I and incidence data for each test substance treated group was compared to each control group.

Reproductive indices:

F0 females: Pregnancy rates, gestation length, percentage of mothers that weaned litters.
F1 females: Mating and fertility indices, pregnancy rates, parturition indices, gestation length, percentage of mothers that weaned litters.

Offspring viability indices:

F1 litters: Mean numbers of live and dead pups at birth, pup survival (at representative intervals through lactation), body w eight, sex distribution.
F2 litters: Mean numbers of live and dead pups at birth, pup survival (at representative intervals through lactation), body w eight, sex distribution.

Clinical signs:

no effects observed

Description (incidence and severity):

No parental animals died or showed clinical signs of toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on body weight/body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There are no comments on this, but as food consumption was not affected, test substance intake should also have not been affected.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry 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

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No abnormal findings.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No effects. A statistically significant reduction in mean gestation length for F2a animals from the 1000 ppm group (22.1 d) versus the pooled F2a controls (22.4 d) was considered spurious by the authors. Also, the lower pregnancy rate recorded in the F1/F2a mating (not significant) was not replicated in the second mating (F1/2b).

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In the highest dose group (F1) there was a non-statistically significant reduction in mean pup weights at birth. There was a slightly lower pup birth weight in mid and high dose groups, but the difference was only statistically significant in the highest dose group compared with the combined controls. There was no difference at other time points or in the F2b generation.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal findings.

Histopathological findings:

not examined

Description (incidence and severity):

VIABILITY (OFFSPRING): The mean numbers of live and dead pups at birth (F1) were considered comparable between the controls and 300 and 1000 ppm groups. In the high dose group there was a decrease in the mean numbers of live pups with a corresponding increase in the mean number of dead pups observed at birth. The differences were not statistically significant compared with the combined control values. The 24 hour and four day survival indices for the treated groups were slightly lower than the control values, and some statistically significant differences from a control group were observed. There was no dose-response relationship so it was concluded that the difference were not treatment-related. There was a slight increase in the mean number of dead pups at birth in the F2a litters at the high dose. However, the increase was attributed to one female that had five dead pups. Pup survival indices were comparable between the groups for the F2 generation.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Reproductive effects observed:

not specified

Table 2 Summary of Gestation length, litter survival, growth of offspring and weaning sex ratio data.

Group (ppm)	Mean gestation period (days)	Mean No.		Mean No. weaned/litter	Litters weaned		Mean live offspring weights (g)				Day 21 sex ratio (M/F)
		Live	Dead		No	%	Day 0	Day 4^a	Day 14	Day 21	No.	Ratio^b
F0 to F1
I/0	22.1	12.3	0.1	9.8	18/18	100	5.9	9.4	27.3	40.8	86/90	0.96
II/0	22.1	11	0.6	8.9	17/18	94.4	5.9	9.3	27.0	41.5	71/81	0.88
I+II	22.1	11.6	0.3	9.4			5.9	9.3	27.2	41.1
III/300	21.9	11.3	0.8	9.5	17/18	94.4	5.9	9.6	27.9	41.3	81/81	1.00
IV/1000	21.9	10.9	0.6	8.7	17/17	100	6.1	9.8	27.3	40.4	65/83	0.78
V/3000	22.3	9.2	1.9	7.9	17/19	89.5	5.7	8.5	26.6	40.8	64/70	0.91
F1 to F2a
I/0	22.4	10.9	0.1	8.7	13/14	92.9	6.3	9.7	26.6	35.4	55/58	0.95
II/0	22.4	10.5	0.3	9.1	14/15	93.3	6.1	10.1	25.5	35.3	64/63	1.02
I+II	22.4	10.7	0.2	8.9			6.2	9.9	26.0	35.3
III/300	22.3	12.4^#	0.1	9.3	17/18	94.4	6.2	9.3	26.0	36.4	83/75	1.11
IV/1000	22.1^#	11.1	0.0	8.8	19/19	100	5.8	9.2	24.8	34.7	84/84	1.00
V/3000	22.2	11.7	0.8	9.5	13/13	100	5.8^#	9.2	25.6	34.6	60/63	0.95
F1 to F2b
I/0	22.3	12.3	0.1	8.9	14/15	93.3	6.0	8.8	26.1	39.2^+	59/66	0.89
II/0	22.1	9.4	0.9	7.8	15/17	88.2	6.1	9.7	29.5	45.0*	61/56	1.09
I+II	22.2	10.8	0.6	8.3			6.0	9.3	27.8
III/300	22.1	12.5	0.3	9.3	19/19	100	6.1	9.5	27.6	41.3	83/93	0.89
IV/1000	22.1	11.4	0.9	9.3	15/17	88.2	5.8	9.0	26.5	39.5 ^++	75/73	1.03
V/3000	22.2	11.1	0.5	9.2	14/15	93.3	5.9	9.3	27.2	40.1	63/66	0.95

Significantly different from control Group I: *p≤0.05; **p≤0.01.

Significantly different from control Group II: +p ≤0.05; ++p ≤0.01.

Significantly different from pooled values of control Groups I and II: #p ≤0.05; ##p ≤0.01.

a Pre-cull weight only.

b Data not analysed statistically.

Conclusions:

In a well conducted and documented, pre-GLP one-generation reproductive toxicity study (reliability score 2), CP 66257 (DTPMP-H) administered via the diet caused no clear treatment-related or statistically significant effects in rats. Pregnancy rate and pup body weight were lower for F2a litters from dams fed diets containing 3000 ppm DTPMP, but this was not replicated in the F1 or F2b litters. A NOAEL of 294 mg/kg bw/d for males and 312 mg/kg bw/d in females was therefore concluded.

Executive summary:

In a well conducted and documented, pre-GLP one generation reproductive toxicity study (reliability score 2), CP 66257 (DTPMP-H) was administered continuously via the diet to Long-Evans rats at concentrations of 300, 1000 and 3000 ppm through one complete generation. Test substance administration to the F0 generation females (20/group) was initiated at the onset of gestation and continued throughout the ensuing gestation and lactation periods. Administration then continued to the F1 generation animals (10 males and 20 females/group) through a growth period and mating, gestation and lactation period for two successive litters. Parameters evaluated included parental mortality, mating-fertility indices, body weight and food consumption data, necropsy observations and organ weight data (absolute and relative) for the F1 parental generation. Litter parameters included gestation length, pup viability at birth and litter survival indices. Pups were evaluated for survival, growth and necropsy observations at weaning.

In the F0 generation, no treatment-related effects in the low and mid dose groups were evident. In the high dose group, females delivered litters containing fewer live pups and more dead pups (not statistically significant). Pups also had a lower weight at birth (

not statistically significant). No other treatment-related effects were observed. In the F1 generation, no treatment-related effects were evident in the low dose group. In the mid dose group, pup weight at birth was lower than control in the first litters (F2a) only. No such effects were observed in the second litters and no other treatment-related effects were observed during the remainder of the study. Gross pathological examination of five adult F1 generation males and females of the control and high dose groups did not reveal any abnormal findings. A NOAEL of 294 mg/kg bw/d for males and 312 mg/kg bw/d in females was therefore concluded.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

294 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no available reproductive toxicity data for DTPMP-xNH4 for the reproductive toxicity endpoint. Therefore, data were read across from the category member DTPMP-H.

The key reproductive study is a one-generation reproductive study (BioDynamics Inc., 1979) is available, in which DTPMP-H was administered continuously via the diet to Long-Evans rats at concentrations of 300, 1000 and 3000 ppm through one complete generation. Test substance administration to the F0 generation females (20/group) was initiated at the onset of gestation and continued throughout the ensuing gestation and lactation periods. Administration then continued to the F1 generation animals (10 males and 20 females/group) through a growth period and mating, gestation and lactation period for two successive litters. The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline. It was not compliant with GLP. In the F0 generation, no treatment-related effects in the low and mid dose groups were evident. In the high dose group, females delivered litters containing fewer live pups and more dead pups (not statistically significant) resulting in a lower live birth index. Pups also had a lower weight at birth (not statistically significant). No other treatment-related effects were observed. In the F1 generation, no treatment-related effects were evident in the low dose group. No such effects were observed in the second litters and no other treatment-related effects were observed during the remainder of the study. Gross pathological examination of five adult F1 generation males and females of the control and high dose groups did not reveal any abnormal findings. A NOAEL of 3000 ppm, equivalent to 294 mg/kg bw/d for males and 312 mg/kg bw/d for females, was therefore concluded.

Effects on developmental toxicity

Description of key information

There are no prenatal developmental toxicity data available for DTPMP-xNH4. Therefore, data were read across from the category member DTPMP-7Na.

 

There is a good quality prenatal developmental toxicity study on DTPMP-7Na (Monsanto, 1982) in which mated female Sprague-Dawley rats (25/dose) were dosed by gavage at doses of 500, 1000, 2000 mg/ kg bw/day (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 post-implantation 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 at least 2000 mg/kg bw/day (active acid).

 

According to ECHA decision number CCH-D-2ii4495836-29-01/F, a prenatal developmental toxicity study (rabbit) with the registered substance DTPMP 5-7 Na-salt is currently being conducted according to OECD 414 and in compliance with GLP. The test is being conducted for a category member that is registered at Annex X. When the study is completed, the registered substance will be updated accordingly.

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

read-across: supporting information

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): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: None, animals were recieved 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:

No data

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

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 fetuses) 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 fetuses or litters with abnormalities and variants) were analysed, when appropriate, with Fisher's exact test and the chi-square test.

Indices:

None

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No deaths occurred prior to the scheduled sacrifice. 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. 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 bodyw eights without uterine contents: 265.8/264.5/260.1/257.2). There was no effect on pregnancy rate or the mean number of corpora lutea. There were no treatment-related findings in the gross necropsy.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no significant differences between any of the treatment groups and the control group in the number of pre- or postimplantation losses, resorptions or mean number of live fetuses. There were no effects on fetal body weight, sex ratios (males/litter: 5.6/5.8/5.9/4.8. Females/litter:  5.9/6.5/5.4/6.4). There were no treatment-related gross external malformations present. 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). The occurence of one female fetus 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.
Skeletal examination revealed single incidences of dwarfism (one female fetus of 500 mg/kg bw/day group) and fused sternebrae (one female fetus of the 2000 mg/kg bw/day group), which were considered spontaneous. Two fetuses from different litters in the 2000 mg/kg bw/day group and one fetus 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.

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 a good quality prenatal developmental toxicity screening study (reliability score 2) conducted using a protocol similar to OECD 414, and to GLP, clear maternal toxicity (approx. 30% decrease in body weight gain, soft stools) was noted in pregnant SD rats given 2000 mg/kg bw/day DTPMP-7Na (Dequest 2061; expressed as active acid) on GD 6-19 (NOAEL 1000 mg/kg bwt/d). The NOAEL for developmental toxicity was 2000 mg/kg bw/day (active acid).

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 fetal weight at any dose. Vertebral anomalies were observed in single fetuses 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 available for DTPMP-xNH4. Therefore, data were read across from the category member DTPMP-7Na.

In a good quality prenatal developmental toxicity screening study on DTPMP-7Na (Monsanto, 1982) mated female Sprague-Dawley rats (25/dose) were dosed by gavage at doses of 500, 1000, 2000 mg/ kg bw/day (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 post-implantation 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 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 at least 2000 mg/kg bw/day (active acid).

Information is available on DTPMP-H from a one-generation reproductive toxicity study (Biodynamics, 1979b), in which DTPMP-H was administered continuously via the diet to Long-Evans rats at concentrations of 300, 1000 and 3000 ppm through one complete generation. Test substance administration to the F0 generation females (20/group) was initiated at the onset of gestation and continued throughout the ensuing gestation and lactation periods. Administration then continued to the F1 generation animals (10 males and 20 females/group) through a growth period and mating, gestation and lactation period for two successive litters. The study was conducted according to a test protocol that is comparable to the appropriate OECD (415) test guideline. It was not compliant with GLP. In the F0 generation, no treatment-related effects in the low and mid dose groups were evident. In the high dose group, females delivered litters containing fewer live pups and more dead pups (not statistically significant) resulting in a lower live birth index. Pups also had a lower weight at birth (not statistically significant). No other treatment-related effects were observed. In the F1 generation, no treatment-related effects were evident in the low dose group. No such effects were observed in the second litters and no other treatment-related effects were observed during the remainder of the study. Gross pathological examination of five adult F1 generation males and females of the control and high dose groups did not reveal any abnormal findings. A NOAEL of 3000 ppm, equivalent to 294 mg/kg bw/d for males and 312 mg/kg bw/d for females, was therefore concluded.

Justification for classification or non-classification

Based on the available data, no classification for reproductive and developmental toxicity is required for DTPMP-xNH4 according to Regulation (EC) No 1272/2008.