Sodium salts of [[(phosphonomethyl)imino]bis[ethane-2,1-diylnitrilobis(methylene)]]tetrakisphosphonic acid (1-3 Na:1)

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12.09. 1977 to 19.06.1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Materials and methods

GLP compliance:

no

Limit test:

no

Test material

Test animals

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): Purina laboratory chow 5001, 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

Administration / exposure

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.
F1 Females
-1M to 2F were caged together for 15 nights or until a sign of mating (sperm/vaginal plug) was observed. Care was taken to avoid brother sister mating. The day on which evidence of mating was observed was defined as 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 F1 males 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.

Doses / concentrationsopen allclose all

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

Examinations

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
-F0 generation: All females sacrificed after weaning of F1 offspring. Necropsy performed.
F1 generation: All surviving males and females sacrificed after weaning and necropsy of the last F2b litter. Necropsy performed. Select organs weighed and tissues performed.
-Extra F1 males and females: Sacrificed at initiation of mating, necropsy performed and discarded.
-Dead or moribund dams: Necropsy performed. Uterine contents examined and presence of implantation sites and/or scars recorded.
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning. Necropsy performed including internal sex determination performed, then carcasses discarded.
GROSS NECROPSY
-Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

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

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Physical observation data by type and incidence were comparable between the control and treated groups throughout gestation and lactation periods.

Mortality:

no mortality observed

Description (incidence):

No parental animals died during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on body weight/body weight gain. Body weights were comparable to the control groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on food consumption. 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 treatment effects.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Pregnancy rates were comparable between the control and treated groups. Mean gestation length was comparable between the control and treated groups. The mean numbers of live and dead pups at birth were considered comparable between the controls and treated Groups III and IV (300 and 1000 ppm, respectively). In Group V (3000 ppm) a decrease in the mean numbers of live pups with a corresponding increase in the mean number of dead pups was observed at birth; however, neither of these values differed statistically from combined control values. The percentage of females that weaned litters was comparable between the control and treated groups.

Effect levels (P0)

Target system / organ toxicity (P0)

Results: P1 (second parental generation)

Target system / organ toxicity (P1)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment effect was indicated in physical observation data

Mortality / viability:

no mortality observed

Description (incidence and severity):

No mortality was observed in the control or treated groups during the growth, rest, gestation, or lactation intervals.
Live birth indices were considered comparable between the control and treated groups 3 and 4. In group 5 (3000 ppm) the live birth index was significantly lower than the incidence data for either control groups. The 24 hour and 4 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. No obvious dose relationship was apparent in the 24 hour and 4 day pup survival indices and no treatment effect was indicated. Pup survival indices for the 14 day and 21 day intervals were generally within the range of values observed for the two control groups and no treatment effect was indicated.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight data during the F1 growth and rest periods were comparable between the control and treated groups,.
Mean pup body weights for groups 3 and 4 were comparable to the controls. In group 5 however, the mean pup weight at birth was slightly lower than the control; this difference was not statistically significant and for the remainder of the lactation period pup weights in group 5 were considered comparable to the control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment effect on food consumption was observed.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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

Other effects:

no effects observed

Description (incidence and severity):

Maternal body weight for the gestation and lactation intervals for the first litter were comparable to the comparable between the control and treated groups. During the gestation and lactation interval for the second litters mean weights were comparable between the control and treated group 3 (300 ppm) and slightly lower than the control data in treated groups 4 and 5 (1000 and 3000 ppm respectively) These differences were not statistically significant.
Mating indices for both males and females were comparable between the control and treated groups during both the first and second mating intervals, similarly fertility indices were comparable between these same groups.
Pregnancy rates were comparable between the control and treated groups 3 and 4 (300 and 1000 ppm respectively) for both mating intervals. In group 5 (3000 ppm) a slight decrease in pregnancy rate was observed during the first mating interval; this difference from the control data was not statistically significant. During the mating interval to produce the second litter (F2b) the pregnancy rate for group 5 was comparable to the control data.
Parturition indices were comparable between the control and treated groups for both mating intervals.
Sex distribution at birth between the control and treated groups were attributed to normal biological variability and no treatment effect was indicated. Sex distribution ratios at day 21 of lactation were similar to ratios observed at day 4 post cull.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Target system / organ toxicity (F1)

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pup survival indices during the lactation period for both F2a and F2b litters were considered comparable between the control and treated groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the first litter mean pup weight at birth was slightly lower than the control values in treated groups 4 and 5 ( 1000 and 3000 ppm respectively). Only in Group 5 was this difference statistically significant from the pooled control data. Mean pup weight at days 4, 14, and 21 were considered comparable between the control and treated groups. In the second litters mean pup weights were considered comparable between control and treated groups throughout lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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:

no effects observed

Description (incidence and severity):

In males the mean terminal body weights were slightly lower that the pooled control value in treated groups 4 and 5 (1000 and 3000 ppm) and higher than control in group 3 (300 ppm). None of these differences were statistically significant. In females, mean terminal body weight were comparable between the control and treated groups.
Sporadic differences from control data were noted for some absolute and relative (to body weight) mean organ weights. These differences were not considered to indicate a test substance related effect.
Organ to brain weight ratios were considered comparable between the control and treated groups for both males and females; no treatment related effect was indicated.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No necropsy and pathology related differences were observed.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Mean gestation length was considered comparable between the control and treated groups for both pregnancy intervals. A statistically significant reduction in mean gestation length in groups 4 (1000 ppm) from the pooled control data for the first pregnancy interval (F2a) was considered spurious and not related to treatment.
The mean numbers of live and dead pups at birth were considered comparable between the control and treated groups for both the first and second litters. A slight increase in the mean number of dead pups at birth in Group 5 for the first litters was attributed to one Group 5 female that had 5 dead pups.
The percentage of females that weaned litters was comparable between the control and treated groups for both the first and second lactation intervals.
Sex distribution ratios for the treated groups both at birth and weaning revealed no treatment effects during either the first or second litters.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Target system / organ toxicity (F2)

Overall reproductive toxicity

Any other information on results incl. tables

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 4a	Day 14	Day 21	No.	Ratiob
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.

Applicant's summary and conclusion

Conclusions:

In the one-generation reproductive toxicity study, conducted prior to OECD Test Guidelines and GLP, no clear treatment-related or statistically significant
effects were seen in rats following DTPMP-H administration via the diet. Pregnancy rate and pup body weight were lower for F2a litters from dams fed diets containing 3000 ppm DTPMP-H, but this was not replicated in the F1 or F2b litters. The concluded NOAEL for reproductive and systemic toxicity was greater than 3000 ppm (equivalent to 294 mg/kg bw/d for males and 312 mg/kg bw/d in females).