Sodium salts of [hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid (4-7 Na:1)

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid

EC Number:

245-781-0

EC Name:

[hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid

Cas Number:

23605-74-5

Molecular formula:

C10H28N2O12P4

IUPAC Name:

[hexane-1,6-diylbis[nitrilobis(methylene)]]tetrakisphosphonic acid

Constituent 2

Reference substance name:

[hexane-1,6-diybis[nitrilobis(methylene)]]tetrakisphosphonic acid

IUPAC Name:

[hexane-1,6-diybis[nitrilobis(methylene)]]tetrakisphosphonic acid

Test material form:

other: white powder

Test animals

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of the test material were mixed in Purina Laboratory Chow® weekly.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow®
- Storage temperature of food: not specified

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test material was given undiluted.

Details on mating procedure:

F0 Females: Females were placed with male rats nightly in a ratio of 2:1. Vaginal smears were taken early in the morning and females were considered to have mated if sperm and/or vaginal plug was observed. The day on which evidence of mating was observed was defined as day 0 of gestation.
F1 Females: One male and two females of equivalent dose levels were caged together nightly for 15 nights or until a sign of mating (sperm and/or 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. F1 females were mated twice and there was a 14-day rest period between weaning of the first litters and initiation of the second mating period.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

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. Dietary administration of the test material continued to the F1 generation animals (10 males and 20 females/group) through a growth period and a mating, gestation and lactation period for two successive litters.

Frequency of treatment:

Continuously, beginning Day 0 of gestation for the F0 females and continued for the F1 males and females through the F2a and F2b litters. (For a total of 281 days)

Details on study schedule:

Test substance administration of the F0 females was initiated the day signs of mating were observed (Day 0 of gestation) and continued throughout the ensuing gestation and lactation periods. F1 offspring were separated from siblings seven days after the weaning (Day 21 of lactation) 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 ensure the required number of adults (10 male 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 re-mated 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.

Litters produced from the mating of the F0 parents were housed together for approximately one week after the last litters had been weaned. Twelve male and twenty-four female offspring were proportionately selected from litters using a random number procedure. The parentage of each selected offspring was recorded to avoid possible brother-sister mating. The F1 generation were housed individually for a growth period of approximately 80 days before being mated to produce the F2a litters.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 ppm

Remarks:

Dose levels based on 100% active ingredient

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Remarks:

Dose levels based on 100% active ingredient

Doses / concentrations 3

Dose / conc.:

3 000 ppm

Remarks:

Dose levels based on 100% active ingredient

No. of animals per sex per dose:

F0 generation females: 20 per group
F1 parents: 10 males and 20 females per group

Control animals:

yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:

BODY WEIGHT:
- Males and non-pregnant Females (F1): body weight was recorded weekly during growth and rest periods of F1 generation.
- Pregnant females (F0, F1 ): recoded on days 0, 6, 15 and 20 of gestation.
- Lactating females (F0, F1): recorded on days 13, 4, 14 and 21 of lactation.

FOOD CONSUMPTION:
- Males and non-pregnant females: recorded weekly during growth and rest periods of F1 generation.

SUBSTANCE CONSUMPTION: Calculated from body weight and food consumption values.
- Males and non-pregnant females: recorded weekly during growth and rest periods of F1 generation.

PHYSICAL OBSERVATIONS:
- FO, F1 parents: For mortality and gross signs of Toxicological or pharmacologic effects in were recorded twice daily. Detailed physical examination for signs of local or systemic toxicity, pharmacological effects and palpation for tissue masses were recorded weekly.

Oestrous cyclicity (parental animals):

Not specified

Sperm parameters (parental animals):

Not specified

Litter observations:

LITTER DATA AND OBSERVATIONS:
- General Appearance and Presence of Dead Pups: examined daily in all litters (F1, F2a, and F2b).
- Live, dead and missing pups counted: on days 0, 1, 4 (pre-cull and post-cull), 14 and 21 of lactation for all litters (F1, F2a & F2b).
- Litters culled: on day 4 of lactation culled to 10 pups (equal number/sex when possible) for all litters (F1, F2a & F2b)
- Live pups as a litter weighed: on days 0, 4 (pre-cull) and 21 (calculated from individual weights) of lactation for all litters (F1, F2a & F2b).
- Live pups weighed individually: on day 21 of lactation for all litters (F1, F2a & F2b).
- External sex determination of pups as a litter: on days 0 and 4 (pre-cull and post-cull) of lactation for all litters (F1, F2a, F2b)
- External sex determination of pups individually: on day 21 of lactation for all litters.

Postmortem examinations (parental animals):

Postmortem:
- F0 generation: All females were sacrificed after weaning of F1 offspring. Necropsy performed for all animals. All abnormal tissues or gross lesions were preserved.
- F1 generation: All surviving males and females were sacrificed after weaning and necropsy of the last F2b litter. Necropsy performed for all animals. Selected organs were weighed and tissues were preserved. Organs were weighed and organ/body weight and organ/brain weight ratios were calculated for F1 parents for brain, pituitary, adrenal (2), testis (2), ovary (2), thyroid (post-fixation, with parathyroids attached), spleen, kidney (2), liver. All abnormal tissues or gross lesions were preserved. Tissues from the following organs were preserved from all F1 survivors: adrenal (2) lymph node, bone (right rib mesenteric junction) pancreas, brain, pituitary, eye (2 with optic nerve) prostate, gonad, spinal cord, testis (2) cervical, ovary (2) spleen, heart stomach, intestine, thymus, cecum, thyroid (with parathyroids), duodenum, urinary bladder, kidney (2) uterus, liver, all gross lesions and tissue masses, lung (2). Histopathological examination was performed for all tissues collected at necropsy for five randomly chosen F1 males and females from the control, (Groups I and II) and high-dose (Group V) groups.
- Dead or moribund dams: Necropsy was performed. Uterine contents were examined and the presence of implantation sites and/or scars was recorded.

Postmortem examinations (offspring):

Offspring:
- Dead pups: Sex was determined and the pups were checked for presence of milk in the stomach and discarded.
- F2a offspring: Necropsy including internal sex determination was performed at weaning. All offspring were then discarded. All abnormal tissues or gross lesions were preserved.
- F2b offspring: The pups were sacrificed at weaning. Necropsy including internal sex determination was performed. Selected tissues were preserved from 10 randomly chosen males and females from all groups. All abnormal tissues or gross lesions were preserved. For 10 randomly selected males and females per group additional tissues were collected: adrenal (2), bone (right rib junction), brain, eye (2 with optic nerve), gonad, testis (2), ovary (2),heart, intestine, cecum, duodenum, kidney ( 2 ), liver. In addition, 10 randomly selected males and females per group at weaning. Lung (2), lymph node mesenteric, pancreas, prostate, spinal cord, cervical, spleen, stomach, thymus, thyroid (with parathyroids), urinary bladder, uterus, all gross lesions and tissue masses.
- F2b offspring found dead: Necropsy including internal sex determination was performed. All offspring were then discarded.
- Culled F1, F2a and F2b offspring: The pups were sacrificed on day 4 of lactation. Necropsy including internal sex determination was performed. All offspring were then discarded.
- F1 offspring not selected as future parents: The pups were sacrificed after weaning and selection of F1 future parents. Necropsy including internal sex determination was performed. All offspring were then discarded.

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 II control values were compared to each other. If no statistically significant differences were noted, control values were combined and values of all test substance-treated groups (III, IV and V ) were compared to the combined control. If Group I and II control values were statistically significant to each other, values of all test substance-treated groups were compared to each control group. 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 (Groups I and II) by the Chi-square test .

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related effects were noted during physical observations.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in the F0 females in eitherthe control or treated groups during the gestation or lactation periods.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight data during gestation and lactation periods were comparable between the control and treated animals.

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Pregnancy rates were comparable between the control and treated animals. Mean gestation length was comparable between the control and treated animals. The mean number of live and dead pups was comparable between the control and treated animals. The percentage of females that weaned litters comparable between the control and treated animals.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

General and reproductive toxicity

Effect level:

>= 3 000 ppm

Based on:

labile/free

Remarks:

acid

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed for males and females.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality was observed in the control or treated Groups III and V (300 and 3000 ppm, respectively). In Group IV (1000 ppm) two females died. Female No. 2004 died during week 1 of the growth period and was replaced with extra female No. 2021 from this same group. Group IV female No. 2021 died on day 22 of gestation for the F2b, pregnancy. This female had delivered four live pups prior to death and necropsy revealed nine term fetuses in utero.

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 animals. Mean body weight data during the two gestation and lactation periods were comparable between the control and treated animals.
Mean terminal body weights for males were comparable between the control and treated groups III and V and significantly lower (p=<0.05) than the pooled control value in Group IV (1000 ppm). Mean terminal body weights for the treated females were considered comparable between the control and treated groups III and IV and slightly lower than control in Group V (3000 ppm).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable between treated and control animals.

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

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):

In the males, mean organ weights, organ/body weight ratios and organ/brain weight ratios were comparable between the control and treated groups. No treatment effect was indicated.
In the females mean thyroid weights (absolute and relative to body weights and brain weights) were significantly lower than the pooled control value for each of the treated groups. Mean spleen weight for group IV females (1000 ppm) was significantly higher than the pooled control value and spleen/body weight ratios were significantly higher than the pooled control group in treated groups IV and V (1000 and 3000 ppm, respectively). Mean spleen/brain weight ratios were comparable between the control and treated groups III and V and significantly higher than the pooled control group in group IV.
All other organ weights and ratios (body weight, brain weight ratios) for the treated group females were considered comparable to control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross necropsy observations of the F1 generation parents and extras not used for the study revealed no findings considered related to test substance administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Gross post-mortem observations and microscopic evaluation of selected tissues from five adult F1 generation males and females of the control (Groups I and II) and high-dose groups indicated no findings considered to be related to the administration of the test substance.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating indices for both males and females were comparable between the control and treated groups during both the first and second mating intervals. Likewise, fertility indices (males) and pregnancy rates were comparable between these same groups.
Parturition indices were comparable between the control and treated groups for both mating intervals.
Mean gestation length for the F2a pregnancies were comparable between the control and treated Groups III and V. In Group IV (1000 ppm) mean gestation length was significantly shorter than the pooled a control group value. The decrease in gestation length in Group IV during the F2a pregnancies was not considered indicative of a treatment effect.
Mean gestation length data for the F2b pregnancies were comparable between the control and treated groups.
In Group III (300 ppm) the mean number of live pups at birth was significantly increased from the pooled control data for the first litters. In Group IV the mean number of live pups at birth was significantly increased from the pooled control values for both the first and second litters. In Group V the mean number of live pups at birth was comparable to the controls for both litters. The increase in mean number of live pups at birth observed in the treated group III and IV was not considered indicative of an adverse effect of treatment.
The mean number of dead pups a t birth was comparable between the control and treated groups for both the first and second litters. The percentage of females that weaned litters was comparable between the control and treated groups for both the first and second lactation intervals.

Effect levels (P1)

1

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

300 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed.

3

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Target system / organ toxicity (P1)

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

other: organ weights; organ weights ratio

Organ:

spleen

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Live birth indices were considered comparable between the control and treated groups. The 24-hour and 4-day survival indices were significantly lower than control in Group III (300 ppm). These differences were attributed to the loss of two entire Group III Litters. Female No. 1804 had 11 pups at birth and no live pups at the 24-hour interval and female No. 1806 had 13 pups at birth and no live pups at the day-4 interval. The 14-day and 21-day survival indices for Group III were comparable to values for control Group I and significantly higher than values for control Group II.
Pup survival indices for treated Groups IV and V were generally comparable to control at each interval during lactation. The statistically significant decrease in 4-day survival index in Group V (3000 ppm) from the control values was attributed to the loss of an entire litter containing 13 pups at birth.
Pup survival during lactation was not considered adversely affected by the treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup weights at days 0, 4, 14, and 21 of lactation were considered comparable between the control and treated groups.

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

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

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 treatment-related effects were noted at necropsy of F1 pups not selected as parents.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex distribution ratios at birth were similar between the control and treated groups. In both treated and control groups sex ddistribution ratios on day 21 (weaning) 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)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The 24-hour and 4-day survival indices for Group III (300 ppm) were lower than control values for the F2a litters. These decreases were attributed to the loss of 12 pups from a single Group III litter during this interval (day 0-4). Pup survival indices for Group III at 14 and 21-days were comparable to control values. Pup survival indices for treated Groups IV and V (1000 and 3000 ppm, respectively) during the F2a lactation period were considered comparable to the control.
During the F2b lactation interval pup survival indices were comparable between the control and treated groups.
No treatment effect on pup survival for either the first or second litters was indicated.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In Group III (300 ppm) mean pup weights throughout the F2a lactation period were lower than the pooled control value; however, only at day 4 was the difference from control statistically significant. Mean pup weight data from Groups IV and V were comparable to the control value throughout the F2a lactation period. In the absence of a dose relationship the decrease in mean pup weights in the low-dose group during the F2a lactation period was not considered treatment-related.
Mean pup weight data during the F2b lactation interval were considered comparable between the control and treated groups.

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

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):

Gross necropsy observations of F2a and F2b offspring revealed no findings considered related to the treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex distribution ratios for the treated groups both at birth, day 4 (post-cull) 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)

1

Key result

Dose descriptor:

NOAEL

Generation:

F2a

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

2

Key result

Dose descriptor:

NOAEL

Generation:

F2b

Effect level:

>= 3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

In the F1 generation, no treatment effects were observed in growth, food consumption, fertility-reproduction or litter examination parameters. At terminal sacrifice, a lower mean body weight was observed in the mid-dose males and high-dose female groups. In the female groups, mean thyroid weights (absolute and relative to body weight and brain weight) were lower in the treated groups and spleen/body weight ratios were higher at the mid - and high- dose levels (only at the mid-dose leve1 was spleen/brain weight ratio increased).

 

No treatment effects on organ weight data were observed in the treated male groups. Gross postmortem observations and evaluation of selected tissues from five adult F1 generation males and females of the control (300 and 1000 ppm) and high-dose (3000 ppm) groups indicated no findings considered related to the administration of the test substance.

In the F1 females the only effect seen was a reduction in the mean thyroid weights. However in the absence of histopathological/clinical pathology examination results, it is not clear whether or not the weight change a real effect.

In the absence of adverse clinical /reproductive effects for this substance it is unlikely to be of toxicological significance.

 

Dose level (ppm)	Mean Weight (g) (Thyroid) F1 Female	Mean Ratio (Thyroid) F1 Female
0	0,018	0,0056
300	0,014	0,0046
1000	0,015	0,0047
3000	0,013	0,0043

Applicant's summary and conclusion

Conclusions:

In a reproductive toxicity study, conducted according to a guideline similar to OECD Test Guideline 416 and prior to GLP, the test substance, HMDTMP-H, was administered continuously via the diet to Long-Evans F0 generation females (20/group) at the onset of gestation and continued throughout the ensuing gestation and lactation periods. Dietary administration was continued to the F1 generation animals (10 males and 20 females /group) through a growth period and a mating, gestation and lactation period for two successive litters.
In the F0 generation, no treatment effects were evident. In the F1 generation, no treatment effects were observed in growth, food consumption, fertility-reproduction or litter examination parameters. In the F1 generation, no treatment effects were observed in growth, food consumption, fertility-reproduction or litter examination parameters. At terminal sacrifice, a lower mean body weight was observed in the mid-dose males and high-dose female groups. In the female groups, mean thyroid weights (absolute and relative to body weight and brain weight) were lower in the treated groups and spleen/body weight ratios were higher at the mid- and high-dose levels; spleen/brain weight ratio was increased only at the mid-dose level. No treatment effects on organ weight data were observed in the treated male groups. In the absence of histopathological/clinical pathology examination results of the thyroid gland, it is not clear whether or not the weight change a real effect. In the absence of adverse clinical /reproductive effects for this substance it is unlikely to be of toxicological significance.
The NOAEL for reproductive toxicity for F0 and F1 was concluded to be greater than 3000 ppm.