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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no reproductive toxicity data for HMDTMP (4-7Na), therefore data are read across from HMDTMP-H (CAS 23605-74-5). See attachment to Section 13 for justification of read-across within HMDTMP Category.

 

In a reproductive toxicity study, conducted according to a guideline similar to OECD Test Guideline 416 and prior to GLP, 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 (Biodynamics, 1979). 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 should be considered an adverse 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 at least 3000 ppm (equivalent to ≥256 mg/kg bw/day active acid).

Link to relevant study records
Reference
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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Long-Evans
Sex:
male/female
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.
Dose / conc.:
300 ppm
Remarks:
Dose levels based on 100% active ingredient
Dose / conc.:
1 000 ppm
Remarks:
Dose levels based on 100% active ingredient
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
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 .
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: 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.
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
Critical effects observed:
no
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: 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.
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
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.
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.
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
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Critical effects observed:
no
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
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
Critical effects observed:
no
Reproductive effects observed:
no

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

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.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
256 mg/kg bw/day
Study duration:
chronic
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 reproductive toxicity data for HMDTMP (4-7Na), therefore data are read across from HMDTMP-H (CAS 23605-74-5). See attached to Section 13 for justification of read-across within HMDTMP Category.

 

In a reasonably well conducted reproductive toxicity study conducted before the adoption of OECD Test Guidelines and pre-dating GLP, the general and reproductive toxicity NOAEL for HMDTMP-H was at least the highest dose tested, 3000 ppm in the diet, in rats (approximately equal to a dose of 256 mg/kg bw/day in males and 297 mg/kg bw/day for females both as active acid) (Biodynamics, 1979). The test protocol followed was similar to the current OECD Test Guideline 416. This study was conducted to assess the long-term effects of HMDTMP-H when 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. Dietary administration of the test substance 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.

 

Parameters evaluated include: parental mortality, mating-fertility indices, body weight and food consumption data, necropsy observations and organ weight data (absolute, relative) for the F1 parental generation. Litter parameters include 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 were evident. 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 level was spleen/brain weight ratio increased). No treatment effects on organ weight data were observed in the treated male groups.

 

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

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity data for HMDTMP (4-7Na) are read-across from the HMDTMP Category member HMDTMP (4-7K). See attachment to Section 13 for justification of read-across within HMDTMP Category.

 

In the key prenatal developmental toxicity study in rats with the Category member HMDTMP (4-7K) (aqueous solution containing 22.6% w/w active acid), conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal and developmental toxicity was concluded to be at least 1000 mg active acid/kg bw/day based on the absence of any treatment-related adverse effects observed at the highest dose tested (Výzkumný ústav organických syntéz a.s., 2021b).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10.03.2020 – 26.10.2020
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:
25th June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
The dose levels of the test item Dequest 2054 (HMDTMP (4-7K)) were recalculated on 100 % of active ingredient HMDTMP-H (Total Active Acid) for the determination of the stability and homogeneity of the application form and main study.
The active ingredient of test item is the acid form, HMDTMP-H (Total Active Acid). The substance has been supplied as a 22.6% w/w Total Active Acid aqueous solution. The water can be considered as an additive. Due to this being an anionised substance, additional water should be used as vehicle. The density of HMDTMP (4-7K) solution is 1.28 g/ml. Therefore, a dose of 1000 mg/kg bw/day of the active ingredient is equivalent to approximately 3.5 ml HMDTMP (4-7K) solution/kg body weight/day.
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
- Age at study initiation: 12 weeks
- Weight at study initiation: not specified
- Fasting period before study: no
- Housing: SPF conditions according to internal SOP No.
Pre-mating period: 2 rats of the same sex in one cage,
Mating period: one male and two females in one cage
Gestation: Pregnant females were housed individually
- Diet (e.g. ad libitum): Complete pelleted diet for rats and mice in SPF breeding
- Water (e.g. ad libitum): Free access to drinking water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Route of administration:
oral: gavage
Vehicle:
other: Aqua pro iniectione
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The application form of the test item, recalculated to 100 % w/w Total Active Acid HMDTMP-H. The application forms (the test item in Aqua pro iniectione) were prepared daily just before administration. The calculated volumes of the test item and vehicle were measured into a glass beaker. The application form was stirred by magnetic stirrer (500 rpm) for 30 minutes and then during the administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Aqua pro iniectione was chosen as vehicle because the substance is anionised and additional water should be used as vehicle.
- Concentration in vehicle: Not specified
- Amount of vehicle (if gavage): The concentrations of all dose levels of HMDTMP-H were adjusted to ensure the administration of 1 mL per 100 g of body weight.
- Lot/batch no. (if required): 2004010227
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and the homogeneity of application form were determined at the test facility by means of measuring the concentrations of K and P (mg/kg) in the test item application form.
Homogeneity of the application form was checked by determination of a concentrations K and P of the test item in three places of the application form (at the bottom, in the middle and on the surface).
Stability of the application form was checked by analyses of the application form within 120 min (at the time 0, 30, 60, 90 and 120 min).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male and 2 females
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from gestation day (GD) 5 to 19
Frequency of treatment:
daily
Duration of test:
20 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle only
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
active acid
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
active acid
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
active acid
No. of animals per sex per dose:
- total number of animals before mating: 100 females and 25 males. Males were not treated and were used only for mating purpose.
- during gestation: 24 probably pregnant females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on no adverse effects in a dose range-finding study in which pregnant female rats were given daily oral gavage doses of 250, 500 and 1000 mg active acid/kg bw/day throughout gestation.
- Rationale for animal assignment (if not random): random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily - during the acclimatization, mating and pregnancy
- Cage side observations checked included: vitality or mortality changes, general health condition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Females were observed in natural conditions in their cages after application, once a day at the similar time each day.

BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: During macroscopy, all orifices, the cranial, thoracic and abdominal cavities were examined. The thyroid glands of control and high dose females were examined at histopathology.

OTHER:
THYROID HORMONE ANALYSIS:
- Time schedule for examinations: on GD 20 prior to termination
- Parameters examined: Blood samples from the pregnant females were assessed for serum levels of thyroid hormones (T3 - Triiodothyronine, T4 - Thyroxine, TSH- Thyroid Stimulating Hormone).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- 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: [one half per litter]
- Skeletal examinations: Yes: [second half per litter]
- Head examinations: Yes: [second half per litter]
- Anogenital distance: Yes [all per litter]
- Sex: Yes [all per litter]
- Individual body weights: Yes [all per litter]
Statistics:
For statistical evaluation the software Statgraphic® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups. The results statistically significant on probability level 0.05 are indicated in the summary tables.

The parametric tests were used for statistical evaluation of:
• body weight of females
• corrected body weight (subtraction weight of uterus from surgery body weight of females)
• food consumption (per interval)
• mean weight of foetuses
• anogenital distance
• thyroid hormones
• biometry of thyroid gland (absolute and relative weight)
• biometry of uterus (absolute and relative weight)
• preimplantation (IUDE) and postimplantation (IUDL) losses

As the first step the test for normality (Shapiro-Wilk test) was performed. If the data were not normally distributed the transformation of data was performed (Box-Cox transformation). If the data were not normal distributed after transformation the non-parametric tests (Kruskal-Wallis Test and Mann-Whitney test) for comparison of the medians were performed.
If data were normally distributed after transformation, the Variance check (Levene’s test) to verify standard deviations within each group was used. One-Way ANOVA (probability level 0.05) was used to detect whether there were any significant differences amongst the means and then the post hoc statistical testing (Fisher's least significant difference - LSD test) for only statistical significant differences was performed.

The non-parametric tests were used for statistical evaluation of following parameters:
• number of corpora lutea, number of implantations, number of resorptions
• number of live foetuses (males, females, both sex)
• number of dead foetuses
The two-groups Mann-Whitney test (probability level 0.05) was applied.

The categorical data (skeletal foetal findings) were analyzed using the generalized linear mixed models with Poisson distribution.
Indices:
Preimplantation loss: Preimplantation loss = [(corpora lutea - implantations) / corpora lutea] x 100
Postimplantation loss: Postimplantation loss = (resorptions/implantations) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were recorded during the treatment period in the control and treated females of all dose levels
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One high dose female died on the 5th day of pregnancy after first administration of the test substance. The cause of death was an intubation error (female No. 196).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weights of treated females at all dose levels were similar to the control group throughout the study. No statistically significant changes were observed. The body weight increments at all dose levels were comparable with the body weight increment of the control females.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The average food consumption of all treated groups was comparable with the control group throughout the study. Statistically significant differences were not detected.
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:
no effects observed
Description (incidence and severity):
The mean absolute and relative weights of uterus at all dose levels were comparable with the control group. Statistically significant differences in uterus biometry were not detected for any of the treated females.
Absolute and relative thyroid gland weights were comparable between the treatment and control females. Statistically significant differences of thyroid weights were not detected.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Uterus dilatation was observed in 2 control group females and 3 middle dose (500 mg active acid/kg bw/day) females. This change is considered to be related to oestrous cycle and since no dose-relationship was observed it is concluded to be not a treatment-related adverse finding. Overall, there were no treatment-related gross pathological findings in any of the treatment group or control group females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological examination of thyroid glands revealed no pathological changes. Because treatment related changes were not recorded for the high dose group, histopathology of thyroid glands at doses of 125 and 500 mg active acid/kg bw/day was not performed.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No statistically significant differences were recorded in serum levels of thyroid hormones T3, T4 and TSH in females from treated groups when compared to control females.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation losses (IUDE) and pos-timplantation losses (IUDL) were comparable between the treatment and the control groups. Statistically significant differences were not detected.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of resorptions were comparable between the treatment and the control groups. Statistically significant differences were not detected.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of resorptions were comparable between the treatment and the control groups. Statistically significant differences were not detected.
Dead fetuses:
no effects observed
Description (incidence and severity):
There was one dead foetus in the middle dose level (500 mg active acid/kg bw/day). Overall, the number of foetuses was comparable between all groups. The average total number of foetuses per litter was comparable across the treated groups and control group.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of females with foetuses on the 20th day of pregnancy was comparable between all treated groups and the control group.
Other effects:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea were comparable between the treatment and the control groups. Statistically significant differences were not detected.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
active acid
Basis for effect level:
other: No treatment-related adverse effects observed
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The foetal mean body weight was slightly decreased in the low dose group (125 mg active acid/kg bw/day) when compared to the control group, without statistical and toxicological significance. Male foetuses were heavier than females in all groups.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The dead foetus at 500 mg active acid/kg bw/day group could not be examined for external malformations due to autolysis. At 500 mg active acid/kg bw/day group, there was one male foetus without a tail and with absent end part of the spine. Since these findings were observed in one foetus from one middle dose litter, they were not considered to be treatment-related and were concluded to be of spontaneous origin. No other external changes were recorded for any of the foetuses.

Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Incomplete ossification of foetal cranium was observed during the examination in all groups, including the control group. The delayed development in parietal bone, interparietal bone, supraoccipital bone, squamous part of temporal bone and arcus zygomaticus was not related to the treatment because the occurence of these findings in litters were high in all groups, including the control group. There were detected statistically significantly increased numbers of foetuses with incomplete ossification of parietal bone, interparietal bone, supraoccipital bone, arcus zygomaticus and squamosal part of temporal bone at 125 mg active acid/kg bw/day in comparison with control. The increased proportions of litters with incomplete ossification premaxilla, maxilla and frontal bone were also detected at the lowest dose level compared to the control, but without statistical significance. These findings in the lowest dose level were without toxicological significance. The litters with holes in the supraoccipital bone were also detected in high percentage in all treated groups as well as in the control group. Other changes of foetal cranium were found only sporadically.
Incomplete ossification of ossification sites of sternebra and unossified ossification sites of sternebra were recorded in foetuses of all treated groups as well as in the control group. It is a normal variability in the schedule of ossification, ossification of the sternum have not to be complete by the 20th day of gestation. These findings were not related to the treatment, because the occurence of these findings was high also in the control litters and dose dependence for these findings was not recorded.
Examination of vertebrae revealed dumbbell and bipartite ossification of vertebrae thoracic centrum in all test groups including control group. The proportions of litters with bipartite ossification of vertebrae thoracic centrum were comparable in treated groups with control group. The proportions of litters with dumbbell ossification of vertebrae thoracic centrum were slightly increased in treated groups in comparison with control group (68.42 % – 84.21 % – 73.68 % – 89.47 %), without statistical significance and dose dependence. The asymmetric ossification of vertebrae thoracic centrum was recorded sporadically in foetuses.The dumbbell ossification of vertebrae lumbar centrum was detected only in litters at treated groups (0 % – 15.79 % – 15.79 % – 15.79 %), but this finding was recorded in relatively low number of foetuses in the treated groups (0 – 4 – 3 – 3). The occurence of this finding was sporadic. Therefore, a dose-response relationship for the findings relating to the vertebrae was not evident.
The malformations, misshapen centrum of lumbar vertebrae and absent centrums of lumbar and sacral vertebrae, were recorded only in one foetus at the dose level 500 mg active acid/kg bw/day (in litter from female No.168 – male foetus No.1). These malformations observed in this one litter in one female were not treatment related and were probably of spontaneous origin.
The changes such as wavy ribs and ribs-supernumerary site were detected also in all groups, including the control group. The proportion of litters with ribs-supernumerary site was slightly increased in the mid dose group in comparison with the control (57.89 % – 63.16 % – 78.95 % – 63.16 %). The proportions of litters with wavy ribs were increased at all dose levels in comparison with the control group (5.26 % – 47.37 % – 21.05 % – 26.32 %), this increase is not related to the test item as statistical significance was not reached. Furthermore, wavy ribs are considered to be a transient finding in rats. This finding was also quantitatively comparable with findings in the control group of other developmental studies performed at the testing facility. Therefore, occurrence of this variation was not considered to be of adverse nature in this study.
The branched rib and the malformation of full supernumerary rib were recorded in one foetus at the 500 mg active acid/kg bw/day dose level (in litter from female No. 168 – female foetus No.6). These malformations observed in this one litter in one female were not treatment related and were probably of spontaneous origin.
Misshapen scapula was recorded only in one 500 mg active acid/kg bw/day group foetus (litter from female No.168 – female foetus No.5). The incomplete ossification of scapula was recorded at all groups. The proportion of litters with incomplete ossification of scapula was slightly increased at the 125 mg active acid/kg bw/day dose level in comparison with the control (47.37 % – 63.16 % – 42.11 % – 47.37 %), without toxicological significance.
Visceral malformations:
no effects observed
Description (incidence and severity):
No adverse findings were observed in any of the test groups or the control group.
Other effects:
no effects observed
Description (incidence and severity):
The anogenital distance (AGD) and corrected AGD of male and female foetuses at all dose levels were comparable with control group.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Remarks:
active acid
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects observed.
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: skull
skeletal: scapule
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
Description (incidence and severity):
- foetal cranium - non-treatment related incomplete ossification at all dose groups and control group; statistically significantly increased number of foetuses with incomplete ossification of parietal bone, interparietal bone, supraoccipital bone, arcus zygomaticus and squamosal part of temporal bone only at 125 mg/kg bw/day, not considered treatment-related; non-statistically significant increased proportions of litters with incomplete ossification of premaxilla, maxilla and frontal bone at 125 mg/kg bw/day and control group.
- sternebra - non-treatment related incomplete ossification at all dose groups and control group.
- vertebrae - non-treatment related dumbbell and bipartite ossification of vertebrae thoracic centrum at all dose groups and control group; sporadic without dose-response relationship dumbbell ossification of vertebrae lumbar centrum at treated groups; non-treatment related and of spontaneour origin misshapen centrum of lumbar vertebrae and absent centrums of lumbar and sacral vertebrae in one 500 mg/kg bw/day foetus.
- ribs - wavy ribs at all dose groups and control group with non-statistically significant increase in the treatment groups when compared to controls; non-treatment related branched rib in one 500 mg/kg bw/day foetus.
- supernumerary ribs - ribs-supernumerary site at all dose groups and control group with non-statistically significant increase in the middle dose group when compared to controls; non-treatment related full supernumerary rib in one 500 mg/kg bw/day foetus.
- scapula - non-treatment related incomplete ossification of scapula at all dose groups and control group; misshapen scapula in one 500 mg/kg bw/day foetus.
Developmental effects observed:
no

Table 1: Pregnancy results

Group

Number of females with foetuses

Number of non-pregnant females**

Number of females without live foetuses but with implantations and resorptions**

0

22

2 (120, 121)

0

125

21

3 (143, 144, 148)

0

500

20

4 (157, 160, 169, 170)

0

1000*

20

3 (188, 192, 198)*

0

Note:  *female No. 196 died on the 5thday of the pregnancy – intubation error

           **numbers in parentheses = individual labels of single animals

           - Only the data from females with live foetuses were used for calculations of means.

 -Data from females with uterus implantations were used for calculation of preimplantation

(IUDE) and postimplantation (IUDL) losses.

Table 2: Body weight in grams (mean ± standard deviation)

Day of pregnancy

Group code
Group code
Group code
Group code

 

0
125
500
1000

1stday

276.2± 13.6

281.3± 16.2

278.6 ± 14.5

281.3 ± 12.8

5thday

294.1 ± 13.1

299.1± 14.7

292.3 ± 10.2

297.2 ± 12.2

8thday

305.1 ± 14.5

309.2 ± 15.5

303.0 ± 13.1

307.8 ± 14.2

11thday

322.4 ± 16.3

325.2 ± 16.6

318.3 ± 14.8

325.2 ± 14.5

14thday

339.6 ± 16.2

341.9 ± 18.9

335.8 ± 15.6

341.8 ± 14.6

17thday

375.8 ± 23.0

377.4 ± 20.0

372.3 ± 19.8

377.9 ± 18.9

20thday

425.3 ± 29.0

431.3 ± 27.2

425.0 ± 25.1

427.6 ± 25.5

Mean increment

149.1

150.0

146.4

156.0

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 3: Food consumption g/animal/day(mean)

Day of pregnancy

Group code
Group code
Group code
Group code

 

0
125
500
1000

5thday

22.78

23.93

23.06

23.37

8thday

24.91

25.47

25.18

24.49

11thday

26.24

27.20

25.99

25.64

14thday

27.67

28.16

28.59

27.67

17thday

29.12

27.85

29.18

29.01

20thday

30.91

30.58

32.20

31.22

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 4: Health condition control

Week of

pregnancy

Group code
Group code
Group code
Group code

 

0
125
500
1000

1stweek

1

1

1

1

2ndweek

1

1

1

1

3rdweek

1

1

1

1

Note:  1– physiological appearance

Table 5: Clinical observation

Week of

pregnancy

Group code
Group code
Group code
Group code

 

0
125
500
1000

1stweek

1

1

1

1

2ndweek

1

1

1

1

3rdweek

1

1

1

1

Note:  1no clinical signs of intoxication

Table 6: Biometry of uterus(mean± standard deviation)

Parameter
Group code
Group code
Group code
Group code

 

0
125
500
1000

Mean necropsy body weight of females (g)

425.3 ± 29.0

431.3 ± 27.2

425.0 ± 25.1

427.6 ± 25.5

Mean absolute weight of uterus (g)

85.56 ± 21.71

87.99 ± 13.85

87.71 ± 15.34

86.35± 11.27

Mean relative weight of uterus (%)

19.94 ± 4.42

20.37 ± 2.71

20.55 ± 2.99

20.15 ± 1.81

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 7: Body weight - corrected* (mean ± SD)

Parameter
Group code
Group code
Group code
Group code

 

0
125
500
1000

Body weight(g)

339.8 ± 18.5

343.3 ± 22.4

337.2 ± 16.7

341.2 ± 18.1

Note:  Statistically significant differences on probability level 0.05 were not detected.

           *body weight correction = necropsy body weight of female – weight of uterus

Table 8: Macroscopic findings(number of females with pathological findings)

Parameter

Group code
Group code
Group code
Group code

 

0
125
500
1000

Number of examined females

24

24

24

24

Number of died females

0

0

0

1*

Without pathological findings

24

24

24

23

Uterus: dilatation

2

0

3

0

Note:  Uterus dilatation = probablynon-pathological finding

           *Female No. 196 – died on the 5thday of the pregnancy – intubation error

Table 9: Parameters of reproduction(number per female, mean ± SD)

Parameter

Group code
Group code
Group code
Group code

 

0
125
500
1000

implantations

15.32± 3.27

16.81± 1.81

16.00± 2.68

16.05± 1.39

resorptions

0.59± 1.14

0.52± 0.87

0.75± 1.68

0.50± 0.76

corpora lutea

16.05± 2.06

17.24± 1.81

16.65± 1.90

16.75± 1.45

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 10: IUDE and IUDL (% per female, mean ± SD)

Parameter

Group Code
Group Code
Group Code
Group Code

 

0
125
500
1000

IUDE

5.20± 14.63

2.44± 3.72

4.27± 9.72

4.05± 4.96

IUDL

3.65± 7.17

3.18± 5.24

4.43± 9.45

3.05± 4.65

Note:  Statistically significant differences on probability level 0.05 were not detected.

The mean of preimplantation and postimplantation losses were calculated from individual data of females.

IUDE = Preimplantation losses

IUDL = Postimplantation losses

Table 11: Weight of thyroid gland (group mean±SD)

Thyroid gland

Group code
Group code
Group code
Group code

 

0

125

500

1000

Absolute weight (g)

0.0312 ± 0.0015

0.0311 ± 0.0015

0.0308 ± 0.0014

0.0312 ± 0.0014

Relative weight (%)

0.0074 ± 0.0006

0.0072 ± 0.0006

0.0073 ± 0.0006

0.0073 ± 0.0005

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 12: Pregnant females - Thyroid hormones (mean concentration ± SD)

Hormone

Group code

Group code

Group code

Group code

 

0

125

500

1000

T3(ng/mL)

0.814 ± 0.109

0.815 ± 0.077

0.780 ± 0.086

0.798 ± 0.088

T4(µg%)

3.407 ± 0.560

3.294 ± 0.561

3.395 ± 0.713

3.276 ± 0.496

TSH(pg/mL)

1016.6 ± 365.2

974.1 ± 360.7

934.6 ± 332.0

992.1 ± 341.1

  Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 13: Number of foetuses (total in group)

Parameter

Group code
Group code
Group code
Group code

 

0
125
500
1000

Total number of live foetuses

324

342

305

311

Number of live foetuses – males

167

165

141

143

Number of live foetuses – females

157

177

164

168

Number of dead foetuses

0

0

1

0

 

Table 14: Number of foetuses (average per litter; mean ± SD)

Parameter

Group code
Group code
Group code
Group code

 

0
125
500
1000

Total number of live foetuses

14.73 ± 3.27

16.29 ± 2.05

15.25 ± 2.84

15.55 ± 1.43

Number of live foetuses – males

7.59 ± 2.75

7.86 ± 2.22

7.05 ± 2.19

7.15 ± 2.32

Number of live foetuses – females

7.14 ± 2.34

8.43 ± 2.68

8.20 ± 2.44

8.40 ± 1.90

Number of dead foetuses

0.00 ± 0.00

0.00 ± 0.00

0.05 ± 0.22

0.00 ± 0.00

Note:  Statistically significant differences on probability level 0.05 were not detected.

 

Table 15: Body weight of foetuses (grams, mean ± SD)

Parameter
Group code
Group code
Group code
Group code

 

0
125
500
1000

weight of all foetues

3.82± 0.82

3.45± 0.49

3.71± 0.64

3.55± 0.50

weight of male foetus

3.90± 0.83

3.54± 0.51

3.85 ± 0.64

3.65± 0.52

weight of female foetus

3.73±0.82

3.35± 0.48

3.60 ± 0.64

3.46± 0.50

Note:  Statistically significant differences on probability level 0.05 were not detected.

  

Table 16: Mean anogenital distance of foetuses (mm)

Group code

0

0

125

125

500

500

1000

1000

Sex

Male

Female

Male

Female

Male

Female

Male

Female

Mean AGD (mm)

3.45

2.15

3.43

2.19

3.56

2.16

3.47

2.18

Corrected AGD (mm)

2.20

1.40

2.25

1.47

2.28

1.42

2.26

1.45

Note: Statistically significant differences on probability level 0.05 were not detected.

  

Table 17: External alterations - foetuses

Alteration

Group code

Group code

Group code

Group code

 

0
125
500
1000

Total number of examined foetuses

324

342

306

311

Total number of examined litters

22

21

20

20

Dead foetus

0

0

1

0

Foetus without tail, absent end part of the spine

0

0

1

0

Number of examined foetuses per litter

(mean ± SD)

14.73

±3.27

16.29

±2.05

15.30

±2.81

15.55

±1.43

Total number of foetuses with alteration

0

0

2

0

Number of foetuses with alteration per litter(mean ± SD)

0.00

±0.00

0.00

±0.00

0.10

±0.31

0.00

±0.00

Proportion of foetuses with alteration per litter

(% mean ± SD)

0.00

±0.00

0.00

±0.00

0.67

±2.07

0.00

±0.00

 

Table 18: Macroscopic changes of soft tissues – individual foetuses

Alteration
Group code
Group code
Group code
Group code

 

0
125
500
1000

Total number of examined foetuses

153

160

141

145

Total number of examined litters

22

21

20

20

With pathological findings - total(number of affected foetuses)

0

0

0

0

Number of examined foetuses in litter

(mean ± SD)

6.95

±1.79

7.62

±1.02

7.05

±1.32

7.25

±0.85

Total number of foetuses with alteration

0

0

0

0

Number of foetuses with alteration in litter

(mean ± SD)

0.00

±0.00

0.00

±0.00

0.00

±0.00

0.00

±0.00

Proportion of foetuses with alteration in litter

(% mean ± SD)

0.00

±0.00

0.00

±0.00

0.00

±0.00

0.00

±0.00

Table 19: Skeletal alterations (number of affected foetuses)

Group code

 

0

125

500

1000

Number of examined foetuses

 

150

162

156

150

CRANIUM

 

 

 

 

 

Nasal bone – i.o.

V

2

0

0

0

Premaxilla – i.o.

V

1

9

0

0

Maxilla – i.o.

V

0

7

0

1

Frontal bone – i.o.

T

6

15

4

5

Parietal bone – i.o.

V

21

74

45

37

Interparietal bonei.o.

V

64

104

87

60

Supraoccipital bone – i.o.

V

47

101

68

69

Supraoccipital bone – hole

T

72

80

90

79

Supraoccipital bone – b.o.

T

0

3

0

2

Arcus zygomaticusi.o.

V

29

62

38

40

Squamosal part of temporal bone – i.o.

V

57

103

91

69

Basisphenoidi.o.

V

2

1

5

2

STERNEBRA

 

 

 

 

 

Sternebra – ossification sites – i.o.

V

127

157

142

147

Sternebra – ossification sites – u.

T

40

73

67

77

Sternebra – ossification sites – b.o.

T

1

1

1

2

VERTEBRAE

 

 

 

 

 

Vertebrae thoracic centrum – b.o.

T

4

8

10

7

Vertebrae thoracic centrum – b.o., as.os.

T

2

0

1

2

Vertebrae thoracic centrum – d.o.

T

28

51

40

44

Vertebrae thoracic centrum – d.o., as.os.

T

2

2

5

2

Vertebrae lumbar centrum – b.o.

T

0

2

0

0

Vertebrae lumbar centrum – d.o.

T

0

4

3

3

Vertebrae lumbar centrum – d.o., as.os.

T

0

0

1

0

Vertebrae lumbar centrum – misshapen

M

0

0

1

0

Vertebrae lumbar centrum – absent

M

0

0

1

0

Vertebrae sacral centrum – absent

M

0

0

1

0

RIBS

 

 

 

 

 

Ribs – supernumerary site

T

22

40

31

30

Ribs – supernumerary full rib

M

0

0

1

0

Ribs – wavy

V

1

22

10

7

Ribs – branched

T

0

0

1

0

SCAPULA

 

 

 

 

 

Scapula – i.o

V

22

16

11

16

Scapula – misshapen

T

0

0

1

0

Note:  The results statistically significantly changedon probability level 0.05 were shaded in the summary table No.20.

Table 20: Skeletal alterations(number of affected litters)

 

Group code

 

0

125

500

1000

Number of examined litters

 

19

19

19

19

CRANIUM

 

 

 

 

 

Nasal bone – i.o.

V

2

0

0

0

Premaxilla – i.o.

V

1

6

0

0

Maxilla – i.o.

V

0

5

0

1

Frontal bone – i.o.

T

3

6

3

3

Parietal bone – i.o.

V

12

15

17

13

Interparietal bonei.o.

V

16

19

18

18

Supraoccipital bone – i.o.

V

16

19

17

18

Supraoccipital bone – hole

T

16

19

19

19

Supraoccipital bone – b.o.

T

0

1

0

1

Arcus zygomaticusi.o.

V

13

18

14

13

Squamosal part of temporal bone – i.o.

V

16

19

18

18

Basisphenoidi.o.

V

2

1

4

2

STERNEBRA

 

 

 

 

 

Sternebra – ossification sites – i.o.

V

19

19

19

19

Sternebra – ossification sites – u.

T

14

18

16

18

Sternebra – ossification sites – b.o.

T

1

1

1

2

VERTEBRAE

 

 

 

 

 

Vertebrae thoracic centrum – b.o.

T

4

6

7

4

Vertebrae thoracic centrum – b.o., as.os.

T

2

0

1

2

Vertebrae thoracic centrum – d.o.

T

13

16

14

17

Vertebrae thoracic centrum – d.o., as.os.

T

2

2

4

2

Vertebrae lumbar centrum – b.o.

T

0

1

0

0

Vertebrae lumbar centrum – d.o.

T

0

3

3

3

Vertebrae lumbar centrum – d.o., as.os.

T

0

0

1

0

Vertebrae lumbar centrum – misshapen

M

0

0

1

0

Vertebrae lumbar centrum – absent

M

0

0

1

0

Vertebrae sacral centrum – absent

M

0

0

1

0

RIBS

 

 

 

 

 

Ribs – supernumerary site

T

11

12

15

12

Ribs – supernumerary full rib

M

0

0

1

0

Ribs – wavy

V

1

9

4

5

Ribs – branched

T

0

0

1

0

SCAPULA

 

 

 

 

 

Scapula – i.o

V

9

12

8

9

Scapula – misshapen

T

0

0

1

0

Note:  Statistically significant differences on probability level 0.05 were not detected.

Table 21: Skeletal alterations(% proportion of litters with affected foetuses)

Group code

 

0

125

500

1000

Number of examined litters

 

19

19

19

19

CRANIUM

 

 

 

 

 

Nasal bone – i.o.

V

10.53

0.00

0.00

0.00

Premaxilla – i.o.

V

5.26

31.58

0.00

0.00

Maxilla – i.o.

V

0.00

26.32

0.00

5.26

Frontal bone – i.o.

T

15.79

31.58

15.79

15.79

Parietal bone – i.o.

V

63.16

78.95

89.47

68.42

Interparietal bonei.o.

V

84.21

100.00

94.74

94.74

Supraoccipital bone – i.o.

V

84.21

100.00

89.47

94.74

Supraoccipital bone – hole

T

84.21

100.00

100.00

100.00

Supraoccipital bone – b.o.

T

0.00

5.26

0.00

5.26

Arcus zygomaticusi.o.

V

68.42

94.74

73.68

68.42

Squamosal part of temporal bone – i.o.

V

84.21

100.00

94.74

94.74

Basisphenoidi.o.

V

10.53

5.26

21.05

10.53

STERNEBRA

 

 

 

 

 

Sternebra – ossification sites – i.o.

V

100.00

100.00

100.00

100.00

Sternebra – ossification sites – u.

T

73.68

94.74

84.21

94.74

Sternebra – ossification sites – b.o.

T

5.26

5.26

5.26

10.53

VERTEBRAE

 

 

 

 

 

Vertebrae thoracic centrum – b.o.

T

21.05

31.58

36.84

21.05

Vertebrae thoracic centrum – b.o., as.os.

T

10.53

0.00

5.26

10.53

Vertebrae thoracic centrum – d.o.

T

68.42

84.21

73.68

89.47

Vertebrae thoracic centrum – d.o., as.os.

T

10.53

10.53

21.05

10.53

Vertebrae lumbar centrum – b.o.

T

0.00

5.26

0.00

0.00

Vertebrae lumbar centrum – d.o.

T

0.00

15.79

15.79

15.79

Vertebrae lumbar centrum – d.o., as.os.

T

0.00

0.00

5.26

0.00

Vertebrae lumbar centrum – misshapenM

M

0.00

0.00

5.26

0.00

Vertebrae lumbar centrum – absentM

M

0.00

0.00

5.26

0.00

Vertebrae sacral centrum – absentM

M

0.00

0.00

5.26

0.00

RIBS

 

 

 

 

 

Ribs – supernumerary site

T

57.89

63.16

78.95

63.16

Ribs – supernumerary full ribM

M

0.00

0.00

5.26

0.00

Ribs – wavy

V

5.26

47.37

21.05

26.32

Ribs – branched

T

0.00

0.00

5.26

0.00

SCAPULA

 

 

 

 

 

Scapula – i.o

V

47.37

63.16

42.11

47.37

Scapula – misshapen

T

0.00

0.00

5.26

0.00

Table 22: Skeletal alterations (% proportion of affected foetuses in litter (mean ± SD))

Group code

 

0

125

500

1000

Number of examined foetuses

 

150

162

156

150

Number of examined litters

 

19

19

19

19

CRANIUM

 

 

 

 

 

Nasal bone – i.o.

V

1.54

± 4.65

0.00

± 0.00

0.00

± 0.00

0.00

± 0.00

Premaxilla – i.o.

V

0.66

 ± 2.87

5.70

± 9.32

0.00

± 0.00

0.00

± 0.00

Maxilla – i.o.

V

0.00

± 0.00

4.46

± 8.20

0.00

± 0.00

0.66

± 2.87

Frontal bone – i.o.

T

4.23

 ± 10.77

9.80

± 15.92

2.56

± 6.58

3.16

± 8.03

Parietal bone – i.o.

V

13.71

 ± 15.67

46.18

± 32.78

28.30

± 18.38

25.51

± 29.69

Interparietal bonei.o.

V

42.02

± 30.21

64.89

± 28.68

55.03

± 28.00

41.37

± 26.57

Supraoccipital bone – i.o.

V

30.39

± 26.16

63.44

± 29.67

42.01

± 25.49

46.97

± 27.47

Supraoccipital bone – hole

T

46.40

± 31.62

49.82

± 21.42

56.90

± 23.30

53.27

± 26.81

Supraoccipital bone – b.o.

T

0.00

± 0.00

1.58

± 6.88

0.00

± 0.00

1.32

± 5.74

Arcus zygomaticusi.o.

V

21.89

± 27.99

38.07

± 21.40

24.36

± 20.77

25.07

± 26.14

Squamosal part of temporal bone – i.o.

V

38.43

 ± 28.10

63.35

± 25.53

57.15

± 29.89

46.84

± 27.99

Basisphenoidi.o.

V

1.24

± 3.73

0.88

± 3.82

3.25

± 6.71

1.46

± 4.48

STERNEBRA

 

 

 

 

 

Sternebra – ossification sites – i.o.

V

85.18

 ± 22.89

96.78

± 11.61

89.35

± 24.11

98.03

± 4.68

Sternebra – ossification sites – u.

T

26.71

 ± 26.31

46.07

± 28.36

42.71

± 30.47

53.02

± 31.49

Sternebra – ossification sites – b.o.

T

0.58

± 2.55

0.48

± 2.09

0.58

± 2.55

1.24

± 3.73

VERTEBRAE

 

 

 

 

 

Vertebrae thoracic centrum – b.o.

T

2.49

± 4.96

4.73

± 9.23

6.78

± 11.31

4.53

± 10.33

Vertebrae thoracic centrum – b.o., as.os.

T

1.24

± 3.73

0.00

± 0.00

0.75

± 3.28

1.32

± 3.94

Vertebrae thoracic centrum – d.o.

T

19.08

 ± 17.59

30.89

± 21.10

25.85

± 20.79

29.81

± 21.07

Vertebrae thoracic centrum – d.o., as.os.

T

1.24

± 3.73

1.06

± 3.20

2.77

± 5.85

1.32

± 3.94

Vertebrae lumbar centrum – b.o.

T

0.00

± 0.00

1.32

± 5.74

0.00

± 0.00

0.00

± 0.00

Vertebrae lumbar centrum – d.o.

T

0.00

± 0.00

2.49

± 6.47

1.94

± 4.65

1.97

± 4.68

Vertebrae lumbar centrum – d.o., as.os.

T

0.00

± 0.00

0.00

± 0.00

0.53

± 2.29

0.00

± 0.00

Vertebrae lumbar centrum – misshapen

M

0.00

± 0.00

0.00

± 0.00

0.58

± 2.55

0.00

± 0.00

Vertebrae lumbar centrum – absent

M

0.00

± 0.00

0.00

± 0.00

0.58

± 2.55

0.00

± 0.00

Vertebrae sacral centrum – absent

M

0.00

± 0.00

0.00

± 0.00

0.58

± 2.55

0.00

± 0.00

RIBS

 

 

 

 

 

Ribs – supernumerary site

T

14.42

± 18.95

24.26

± 31.81

20.62

± 20.32

19.56

± 23.69

Ribs – supernumerary full rib

M

0.00

± 0.00

0.00

± 0.00

0.58

± 2.55

0.00

± 0.00

Ribs – wavy

V

0.58

± 2.55

14.72

± 23.40

6.15

± 12.32

5.65

± 10.50

Ribs – branched

T

0.00

± 0.00

0.00

± 0.00

0.58

± 2.55

0.00

± 0.00

SCAPULA

 

 

 

 

 

Scapula – i.o

V

14.97

± 19.26

10.27

± 10.30

7.05

± 9.67

10.52

± 12.46

Scapula – misshapen

T

0.00

± 0.00

0.00

± 0.00

0.58

± 2.55

0.00

± 0.00

Conclusions:
In the prenatal developmental toxicity study with HMDTPMP (4-7K) (aqueous solution containing 22.6% w/w active acid), conducted according to OECD Test Guideline 414 and in compliance with GLP, the NOAEL for maternal and developmental toxicity was concluded to be greater than 1000 mg active acid/kg bw/day based on no treatment-related adverse effects observed at the highest dose tested.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 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

Prenatal developmental toxicity data for HMDTMP (4-7Na) are read-across from the HMDTMP Category member HMDTMP (4-7K). See attachment to Section 13 for justification of read-across within HMDTMP Category.

 

In the key prenatal developmental toxicity study with the Category member HMDTMP (4-7K) (aqueous solution containing 22.6% w/w active acid), conducted according to OECD Test Guideline 414 and in compliance with GLP, 0, 125, 500 and 1000 mg active acid/kg bw/day in the vehicle aqua pro iniectione were administered daily by oral gavage to pregnant female rats from gestation day 5 to gestation day 20 (Výzkumný ústav organických syntéz a.s., 2021b). The doses selected in the study were based on the absence of adverse effects in a dose range-finding study. The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored throughout the study. On gestation day 20 blood was collected from the pregnant females to determine thyroid hormone levels (T3, T4, TSH). On the same day the maternal animals were euthanised, the uterine contents were examined, and the foetuses were assessed for external malformations, changes on soft tissues and skeleton. The foetuses were weighed and the anogenital distances (AGD) were measured. The thyroid glands from the maternal animals were examined at histopathology.

 

There were no test material-related unscheduled deaths during the study at any dose level. One female in the 1000 mg active acid/kg bw/day group died on the 5th day of pregnancy due to an intubation error.

 

No adverse changes in health condition and no clinical symptoms of toxicity were observed in any of the animals following administration of the test material at any dose level. There were no toxicologically significant treatment-related effects on body weight or food consumption in any of the animals.

 

Evaluation of uterine weights (absolute and relative weight of uterus) did not reveal any toxicologically significant and treatment-related effects.

 

There were no macroscopic changes in the examined organs of the pregnant females. The reproduction parameters such as number of females with live foetuses, number of live and dead foetuses, early and late resorptions and sex ratio of foetuses were unaffected by the treatment with the test material.

 

Examination of the thyroid glands including absolute and relative weight of thyroid gland, histological examination of thyroid gland and serum levels of thyroid hormones did not reveal any changes associated with the application of the test material.

 

There was one dead foetus from litter No. 163 in the middle dose level (500 mg active acid/kg bw/day). Overall, the number of foetuses was comparable between all groups and there was no test material-related foetal mortality at any dose level. The average total number of foetuses per litter was comparable across the treated groups and control group. Detailed necropsy of foetuses did not reveal an increase of external and visceral variations and malformations at any dose level. During the pathological examination of external alterations one foetus without a tail and with absent end part of the spine was recorded for litter No. 168 in the 500 mg active acid/kg bw/day group. The one death and the single instance of external findings were observed in single foetuses from two different litters and were therefore considered to be not treatment-related and probably of spontaneous origin.

 

Based on statistical evaluation of mean values of foetal body weight, no significant growth retardation was detected in treated groups. The foetal body weight was slightly decreased in the low dose group, but without statistical or toxicological significance.

 

The mean AGD and corrected AGD of male and female foetuses in treated groups was not statistically significantly different from the control group.

 

No statistically significant differences with toxicological significance were recorded in number of litters during the statistical evaluation of skeletal findings. Examination of the foetal skeletons indicated mainly delayed development of the skeleton in all groups, including the control group. Malformations such as misshapen centrum of lumbar vertebrae and absent centrums of lumbar and sacral vertebrae, supernumerary full rib were recorded only in two foetuses in one litter at 500 mg active acid/kg bw/day. These malformations were observed in one litter from one female without a dose response relationship. Therefore, they were concluded to be not treatment-related and probably of spontaneous origin.

 

The proportion of litters with wavy ribs was increased at all dose levels in comparison with the control group (5.26 % – 47.37 % – 21.05 % – 26.32 %), without statistical significance and without dose response relationship. Occurrence of this variation was not considered to be of adverse nature.

 

The NOAEL for maternal and developmental toxicity was concluded to be at least 1000 mg active acid/kg bw/day based on the absence of treatment-related adverse effects observed at the highest dose tested.

 

In the dose range finding study with HMDTMP (4-7K) (aqueous solution containing 22.6% w/w active acid), not conducted according to OECD Test Guideline or in compliance with GLP, oral administration of HMDTPMP (4-7K) to pregnant females by gavage from the 5th to the 19th day of pregnancy at the dose levels 250, 500 and 1000 mg active acid/kg bw/day did not cause mortality of pregnant females (Výzkumný ústav organických syntéz a.s., 2020b). No adverse changes of health condition and no clinical symptoms of intoxication were observed in females at any dose level after administration of the test material. The test material did not affect the growth of maternal animals at any dose level. The haematological examination did not show significant differences between treated and control groups. The pathological examination of females and foetuses revealed no serious macroscopic changes. The reproduction parameters – number of implantations and corpora lutea were not significantly changed at any dose levels. Only the number of resorptions was slightly increased at 1000 mg active acid/kg bw/day. On the basis of these results dose levels of 125, 500 and 1000 mg active acid/kg bw/day were selected for the main prenatal developmental toxicity study.

Justification for classification or non-classification

Based on the available read-across data, no classification is required for HMDTMP (4-7Na) for reproductive and developmental toxicity according to Regulation (EC) No 1272/2008.

Additional information

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