Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The key reproductive study is a multigenerational study for the analogous substance, ATMP-H, conducted prior to the adoption of OECD test guidelines and pre-GLP (Biodynamics Inc., 1979a). It is judged to be reliable with restrictions. Male and female Long-Evans rats were administered ATMP-H 60 days prior to mating (F0) and continuously thereafter (F1, F2, F3) in the diet at fixed concentrations of 0, 300, 1000 and 3000 ppm for three consecutive generations. The litters from F0, F1 and F2 matings were raised to maturity and also mated. Offspring from the first litter of each generation (F1a, F2a, F3a) were taken for necropsy on lactation Day 21. The parents were re-mated following a 14-day rest period and the offspring randomly selected at seven days post-weaning to continue as the F1b and F2b generation parents. Remaining F1b and F2b animals, as well as the F3a and F3b generation, were taken for necropsy. A gross internal examination was conducted on these animals. Randomly selected offspring from the F3a litters (10 pups/sex/group) were necropsied and selected tissues examined microscopically. Evaluations of adult mortality, mating, pregnancy, fertility, body weight data, food consumption data (growth and rest periods), litter survival, offspring viability at parturition, offspring weight and sex, and necropsy of adults and offspring, did not indicate any treatment-related adverse effects. The NOAEL for general toxicity and reproductive toxicity was greater than the highest dose tested, 3000 ppm. The concentration of the test substance and mean weekly food intake values were used to determine the approximate doses received by the animals. 3000 ppm was approximately equal to a dose of 275 mg/kg bw/day in males and 310 mg/kg bw/day in females.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29.10.1976 to 15.08.1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Three generation reproduction toxicity study with the following restrictions: no assessment of estrus cycle, sperm parameters, sexual milestones, no analytical confirmation of exposure levels.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Long-Evans
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Blue Spruce Farms, Altamont, New York.
- Age at study initiation: (P) 6-7 weeks
- Weight at study initiation: (P) males approx. 370 g, females approx. 240 g at mating
- Fasting period before study: No data
- Housing: Individually (except during mating and lactation) in elevated stainless steel wire mesh cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days.


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


IN-LIFE DATES: From: 12.11.1976 To: 15.08.1978
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow (standard laboratory diet)
- Storage temperature of food: No data

Details on mating procedure:
- M/F ratio per cage: 1/2 (See table 1)
- Length of cohabitation: Overnight for up to 15 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- Remated (for F1b/F2b/F3b generation) following a 14 d rest period.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged: individually in elevated stainless steel wire mesh cages.
- Any other deviations from standard protocol: None apparent.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet samples were taken from control and treated groups weekly. Samples were stored frozen and sent to the sponsor at regular intervals throughout the study. No further details.
Duration of treatment / exposure:
From 60 days prior to first mating of P generation then continuous over 3 generations . Duration of test in total was approximately 21 months.
Frequency of treatment:
Daily
Details on study schedule:
- F1 and F2 parental animals not mated until after a growth period (unspecified duration) following selection from the F1b and F2b litters.
- Selection of parents from F1 and F2 generation when pups were 7 days post weaning.
- Age at mating of the mated animals in the study: Not clear, but there was a 14 day rest period between matings.
Dose / conc.:
300 ppm (nominal)
Remarks:
nominal in diet
See table 3 for conversion to mg/kg bw/day
Dose / conc.:
1 000 ppm (nominal)
Remarks:
nominal in diet
See table 3 for conversion to mg/kg bw/day
Dose / conc.:
3 000 ppm (nominal)
Remarks:
nominal in diet
See table 3 for conversion to mg/kg bw/day
No. of animals per sex per dose:
12 male and 24 females (see Table 1)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random
Positive control:
None
Parental animals: Observations and examinations:
Examination conducted on F0, F1, F2 AND F3 (all adult generations)

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Gross signs twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during growth and rest periods of all animals. As well as pregnant females (F0, F1b, F2b) on GD 0, 6, 15 and 20 and lactating females (F0, F1) on LD 0, 4, 14 and 21.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly for males and non-pregnant females.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


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


OPHTHALMOSCOPIC EXAMINATION: F0 parents after weaning of F1b litter.
Oestrous cyclicity (parental animals):
Not investigated.
Sperm parameters (parental animals):
Not investigated.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, to 10 pups/sex/litter as nearly as possible; excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioral abnormalities. See Tables 4, 5 and 6 for pup survival data.


GROSS EXAMINATION OF DEAD PUPS:
yes, sex determined and stomach checked for presence of milk. Cause of death was not determined.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after completion of pup selection for the F0 and F1 generations, and after weaning of last litters for the F2 generation.
- Maternal animals: All surviving animals after completion of pup selection for the F0 and F1 generations, and after weaning of last litters for the F2 generation. Also non-pregnant dams from first mating.
- Dead and moribund animals examined as death occurred.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Dams uterine contents examined for the presence of implantation sites and/or scars.


HISTOPATHOLOGY / ORGAN WEIGHTS: None scheduled for adults.Only grossly abnormal tissues were examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1a/F2a/F3a offspring not selected as parental animals were sacrificed at 21 days of age.
- F1b and F2b progeny (non-parental): sacrificed after pup selection for next generation.
- F3b sacrificed at weaning.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:


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


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 2 were prepared for microscopic examination from 10 pups/sex/group of the F3b generation. In addition any grossly abnormal tissues were examined.
Statistics:
Offspring body weights, off-spring numbers (LD 0 LD 4): F-test and Student's T-test.
Offspring survival, litter deaths, litters weaned, mortality, mating rates, pregnancy rates, fertility rates: Chi square.
Body weights, body weight change, food intake: Dunnett's test.
Reproductive indices:
mating indices (%), pregnancy rates (%) and fertility (%). No details given.
Offspring viability indices:
No details given.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F0: One mid dose male was sacrificed in a moribund state during mating for F1b litter ("severely tilted head"). One high dose female was sacrificed post-weaning of F1b litter with "extremely distended abdomen". There was no dose response and the deaths were considered sporadic, therefore not related to treatement. Physical observations comparable between all groups.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The ophthalmoscopic examination revealed four rats (one control female, two mid dose males and one high dose male) with ocular abnormalities. However, these were not considered to be treatment related.
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): One mid dose male was sacrificed in a moribund state during mating for F1b litter ("severely tilted head"). One high dose female was sacrificed post-weaning of F1b litter with "extremely distended abdomen".  There was no dose response and the deaths were considered sporadic, therefore not related to treatment.
Physical observations comparable between all groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): There was no effect in either sex on mean body weight or body weight gain during growth or rest periods. There were no effects on maternal body weight or body weight change during gestation or lactation periods.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): See Table 3


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Gestation length, mean number of live and dead pups at birth and percentage of live pups at birth were comparable between groups. A high number of dead pups within a single mid dose litter lead to a significant decrease in the survival index at birth in the mid dose group for F1b.

GROSS PATHOLOGY (PARENTAL ANIMALS): There were no adverse findings.


HISTOPATHOLOGY (PARENTAL ANIMALS): Not conducted.


OTHER FINDINGS (PARENTAL ANIMALS): The ophthalmoscopic examination revealed four rats (one control female, two mid dose males and one high dose male) with ocular abnormalities. However, these were not considered to be treatment related.
Key result
Dose descriptor:
NOAEL
Remarks:
F0
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:
effects observed, non-treatment-related
Description (incidence and severity):
F1 (parental animals): Severely tilted heads in one control male and one high dose female.
Red and swollen ears on the tagged ear were noted in F1 and F2 adults.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
F1: One mid dose male died during mating. One high dose female died during post-weaning interval, prior to sacrifice. One control male and one high dose female sacrificed with "severely tilted heads" (male sacrificed wk 4, female in rest period between matings). There was no dose response and the deaths were considered sporadic, therefore not related to treatment. Physical observations comparable between all groups.
F2: One low dose female died during wk 3 of the growth period. One mid dose male died during wk 8 of the growth period. One mid dose female died on GD 21 for the F3b litter. One high dose male died during the mating interval to produce the second litter. There was no dose response and the deaths were considered sporadic, therefore not related to treatment. Physical observations comparable between all groups.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): F1: One mid dose male died during mating. One high dose female died during post-weaning interval, prior to sacrifice. One control male and one high dose female sacrificed with "severely tilted heads" (male sacrificed wk 4, female in rest period between matings). F2: One low dose female died during wk 3 of the growth period. One mid dose male died during wk 8 of the growth period. One mid dose female died on GD 21 for the F3b litter. One high dose male died during the mating interval to produce the second litter. There was no dose response and the deaths were considered sporadic, therefore not related to treatment.
Physical observations comparable between all groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): There was no effect in either sex on mean body weight or body weight gain during growth or rest periods. There were no effects on maternal body weight or body weight change during gestation or lactation periods.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): See Table 3


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Gestation length, mean number of live and dead pups at birth and percentage of live pups at birth were comparable between groups. A high number of dead pups within a single mid dose litter lead to a significant decrease in the survival index at birth in the mid dose group for F1b. The F2 generation high dose group had a significantly higher survival index at birth for the second litters. No adverse effect was concluded. There was no effect on mating indices (%), pregnancy rates (%) or fertility (%).


GROSS PATHOLOGY (PARENTAL ANIMALS): There were no adverse findings.


HISTOPATHOLOGY (PARENTAL ANIMALS): Not conducted.


OTHER FINDINGS (PARENTAL ANIMALS): Not conducted
Key result
Dose descriptor:
NOAEL
Remarks:
F1 / P1
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
Remarks:
F2 / P2
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:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING): Significant decrease (P<0.01) in survival index at birth in the second mid dose litter (F1 for F2b). This was not considered indicative of a treatment-related effect by the study authors since comparable changes this was not replicated in other phases of the study, nor was any dose/response relationship present. Survival at birth was significantly increased (P<0.01) for the high dose F3b litter. Some statistically significant differences were apparent in postnatal survival indices between control and treated groups, however these were not considered adverse by the authors since no trend was present.  Comment: these differences generally reflected a statistically significant enhancement in survival relative to the controls (9 instances), while decreased survival was relatively infrequent (2 instances). The percentages of litters with offspring deaths and litters weaned were comparable between control and treated groups.

CLINICAL SIGNS (OFFSPRING): None reported.


BODY WEIGHT (OFFSPRING): Comparable between all groups.


GROSS PATHOLOGY (OFFSPRING): No adverse findings.


HISTOPATHOLOGY (OFFSPRING): No adverse findings.

OTHER LITTER PARAMETERS: Sex ratio was not affected by treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
F1
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:
no mortality observed
Description (incidence and severity):
Significant decrease (P<0.01) in survival index at birth in the second mid dose litter (F1 for F2b). This was not considered indicative of a treatment-related effect by the study authors since comparable changes this was not replicated in other phases of the study, nor was any dose/response relationship present. Survival at birth was significantly increased (P<0.01) for the high dose F3b litter. Some statistically significant differences were apparent in postnatal survival indices between control and treated groups, however these were not considered adverse by the authors since no trend was present.  Comment: these differences generally reflected a statistically significant enhancement in survival relative to the controls (9 instances), while decreased survival was relatively infrequent (2 instances). The percentages of litters with offspring deaths and litters weaned were comparable between control and treated groups.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Scattered red foci present in lung from some F2b offspring from the mid and high dose groups (not present in controls and low dose group) considered unrelated to treatment by authors (since not present in other generations). All other necropsy observations similar for control and treated litters. Evaluation of selected tissues from 10 control weanlings and 10 high dose weanlings from the F3b generation revealed no abnormalities. Changes present in lung consistent with minimal to mild interstital pneumonia, microscopic appearance of the gonads unremarkable and consistent with sexually immature rats.
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING): Significant decrease (P<0.01) in survival index at birth in the second mid dose litter (F1 for F2b). This was not considered indicative of a treatment-related effect by the study authors since comparable changes this was not replicated in other phases of the study, nor was any dose/response relationship present. Survival at birth was significantly increased (P<0.01) for the high dose F3b litter. Some statistically significant differences were apparent in postnatal survival indices between control and treated groups, however these were not considered adverse by the authors since no trend was present.  Comment: these differences generally reflected a statistically significant enhancement in survival relative to the controls (9 instances), while decreased survival was relatively infrequent (2 instances). The percentages of litters with offspring deaths and litters weaned were comparable between control and treated groups.

CLINICAL SIGNS (OFFSPRING): None reported.


BODY WEIGHT (OFFSPRING): Comparable between all groups.


GROSS PATHOLOGY (OFFSPRING): scattered red foci present in lung from some F2b offspring from the mid and high dose groups (not present in controls and low dose group) considered unrelated to treatment by authors (since not present in other generations). All other necropsy observations similar for control and treated litters. Evaluation of selected tissues from 10 control weanlings and 10 high dose weanlings from the F3b generation revealed no abnormalities. Changes present in lung consistent with minimal to mild interstital pneumonia, microscopic appearance of the gonads unremarkable and consistent with sexually immature rats.


HISTOPATHOLOGY (OFFSPRING): No adverse findings.

OTHER LITTER PARAMETERS: Sex ratio was not affected by treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
F2
Generation:
F2
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
Remarks:
F3
Generation:
other: F3
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


Table 3 Test substance intake based on food intake (weekly mean data) measurements .

    Males (mg/kg bw/day)   Males (mg/kg bw/day)        Males (mg/kg bw/day)   Females (mg/kg bw/day)   Females (mg/kg bw/day)        Females  (mg/kg bw/day)
 Group (ppm)  II (300)  III (1000)  IV (3000)  II (300)  III (1000)  IV (3000)
 F0 growth  33.4  111.6  342.3  37.3  117.1  362.5
 F0 rest  18.4  60.7  182.9  24.3  75.7  247.2
 F1 growth  26.3  89.6  270.2  28.1  98.4  290.2
 F1 rest  14.4  41.7  141.0  20.1  67.3  201.1
 F2 growth  29.6  95.4  296.6  34.3  112.6  337.8
 F2 rest  17.6  58.3  171.9  25.0  81.4  243.9



Table 4 Summary of offspring survival for the F1 generation.

 Group (ppm)  Mean gestation length  % pups born alive  Mean no weaned/litter  Postnatal survival (%)      Postnatal survival (%)      Postnatal survival (%)        % litters with death (days 0 -21)c % litters weanedc  Sex ratio (M/F) 
         0 -4a  4 -14b  14 -21      
   F0 to F1a                            
 I (0)  22.5  98.8  9.1  92.0  94.3  100  57.1  95.2  0.87
 II (300)  22.4  98.2  8.6  97.3*  90.5  100  65  100  0.87
 III (1000)  22.5 98.1  7.6*   96.2 85.4**   100  47.6 95.2   0.88
 IV (3000)  22.3  98.1  8.7  99.2**  91.4  99  52.2 100   1.24
    F0 to F1b                                           
 I (0)  22.1  93.0  8.9  87.2  96.0  99.3  61.1  88.9  1.07
 II (300)  22.1  96.0  8.7  91.7  91.3  99.4  83.3  100  1.12
 III (1000)  22.1  97.2  9.3  94.8*  93.7  100  56.3 100   0.84
 IV (3000)  22.1  95.8 9.2   97.6**  96.5  99.5  28.6  100  0.96

Significantly different from control *p0.05; **p0.01

aComparison between days for postnatal offspring survival are calculated using Day 4 pre-cull data.

bComparison between days for postnatal offspring survival are calculated using Day 4 post-cull data.

cOnly those pups found alive at Day 0 of lactation are used in calculations.

Table 5 Summary of offspring survival for the F2 generation.

 Group (ppm)  Mean gestation length  % pups born alive  Mean no weaned/litter  Postnatal survival (%)    Postnatal survival (%)    Postnatal survival (%)        % litters with death (days 0 -21)c % litters weanedc  Sex ratio (M/F) 
         0 -4a  4 -14b  14 -21      
     F1 to F2a                                          
 I (0)  22.2  99.5  9.2  96.2  93.5  100  33.3  94.4 1.09
 II (300)  22.3  100  9.5  96.8  99.5**  100  35.0  100 1.18
 III (1000) 22.1  99.6 9.4 95.4  99.5**  98.6  40.9  100 1.06 
 IV (3000)  22.3 97.4  9.1 97.3   100**  100  30.4 100  0.92
    F1 to F2b                                           
 I (0) 22.4  99.3  8.8  78.5  100  100 35.7  85.7 0.89
 II (300)  22.1  96.1 8.8  93.6**  92.5  98.1  61.1 100  1.11
 III (1000)  22.1  92.5** 8.2  92.4**  88.5** 58.8   93.8 93.8  0.95
 IV (3000)  22.5  99.1 9.0  96.6**  97.8  98.9  50.0  100  0.94

Significantly different from control *p0.05; **p0.01

aComparison between days for postnatal offspring survival are calculated using Day 4 pre-cull data.

bComparison between days for postnatal offspring survival are calculated using Day 4 post-cull data.

cOnly those pups found alive at Day 0 of lactation are used in calculations.

Table 6 Summary of offspring survival for the F3 generation.

 Group (ppm)  Mean gestation length  % pups born alive  Mean no weaned/litter   Postnatal survival (%)   Postnatal survival (%)    Postnatal survival (%)        % litters with death (days 0 -21)c % litters weanedc  Sex ratio (M/F) 
         0 -4a  4 -14b  14 -21      
   F2 to F3a                                           
 I (0)  22.3  97.9  7.8  89.5  94.3  99.4  54.5  95.5 0.91
 II (300)  22.4  91.3  6.4 87.2  89.5  100  75.0  100 1.13
 III (1000) 22.2 96.2 7.7 85.9 89  100  57.1  87.7 1.09
 IV (3000) 22.2  98.6  8.8 96.7**  91.9  99.4  66.7 94.7 1.11 
  F2 to F3b                                             
 I (0) 22.2  92.9  9.1  89.5  97.4  98.6 50.0  88.9 1.00
 II (300)  22.4 96.1 8.7  92.4  98.5  99.2  43.8 93.8  1.06
 III (1000)  22.1  92.7 8.3  93.5  97.3 100  38.5 100  0.83
 IV (3000) 22.2   98.5** 9.2  89.9  98.7  100  52.9  94.1 1.45 

Significantly different from control *p0.05; **p0.01

aComparison between days for postnatal offspring survival are calculated using Day 4 pre-cull data.

bComparison between days for postnatal offspring survival are calculated using Day 4 post-cull data.

cOnly those pups found alive at Day 0 of lactation are used in calculations.




Conclusions:
In a reasonably well conducted three generation reproductive toxicity study conducted before the adoption of OECD test guidelines and GLP, the general and reproductive toxicity NOAEL for ATMP was greater than the highest dose tested, 3000 ppm in the diet, in rats (approximately equal to a dose of 275 mg/kg bw/day in males and 310 mg/kg bw/day for females).
Executive summary:

Male and female Long-Evans rats were administered CP 42902 (nitrilotrimethylenetris(phosphonic acid)) continuously in the diet at fixed concentrations of 0, 300, 1000 and 3000 ppm for three consecutive generations. The litters from F0, F1 and F2 matings were raised to maturity and also mated. Offspring from the first litter of each generation (F1a, F2a, F3a) were taken for necropsy on lactation Day 21. The parents were remated following a 14 d rest period and the offspring randomly selected at seven days post-weaning to continue as the F1b and F2b generation parents. Remaining F1b and F2b animals, as well as the F3a and F3b generation, were taken for necropsy. A gross internal examination was conducted on these animals. Randomly selected offspring from the F3a litters (10 pups/sex/group) were necropsied and selected tissues examined microscopically. Evaluations of adult mortality, mating, pregnancy, fertility, body weight data, food consumption data (growth and rest periods), litter survival, offspring viability at parturition, offspring weight and sex, and necropsy of adults and offspring, did not indicate any treatment related adverse effects. The NOAEL for general toxicity and reproductive toxicity was greater than the highest dose tested, 3000 ppm. The concentration of the test substance and mean weekly food intake values were used to determine the approximate doses received by the animals. 3000 ppm was approximately equal to a dose of 275 mg/kg bw/day in males and 310 mg/kg bw/day in females.

Endpoint:
three-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attachment for justification of read-across. ATMP acid is a member of the ATMP phosphonates category, structurally related to the DTPMP category.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across: supporting information
Key result
Dose descriptor:
NOAEL
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
Remarks:
F1
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
Remarks:
F2
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:
F1
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:
F2
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:
other: F3
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Reproductive effects observed:
no
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
292 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The key reproductive study is a multigenerational study for the analogous substance, ATMP-H, conducted prior to the adoption of OECD test guidelines and pre-GLP (Biodynamics Inc., 1979a). It is judged to be reliable with restrictions. Male and female Long-Evans rats were administered ATMP-H 60 days prior to mating (F0) and continuously thereafter (F1, F2, F3) in the diet at fixed concentrations of 0, 300, 1000 and 3000 ppm for three consecutive generations. The litters from F0, F1 and F2 matings were raised to maturity and also mated. Offspring from the first litter of each generation (F1a, F2a, F3a) were taken for necropsy on lactation Day 21. The parents were re-mated following a 14-day rest period and the offspring randomly selected at seven days post-weaning to continue as the F1b and F2b generation parents. Remaining F1b and F2b animals, as well as the F3a and F3b generation, were taken for necropsy. A gross internal examination was conducted on these animals. Randomly selected offspring from the F3a litters (10 pups/sex/group) were necropsied and selected tissues examined microscopically. Evaluations of adult mortality, mating, pregnancy, fertility, body weight data, food consumption data (growth and rest periods), litter survival, offspring viability at parturition, offspring weight and sex, and necropsy of adults and offspring, did not indicate any treatment-related adverse effects. The NOAEL for general toxicity and reproductive toxicity was greater than the highest dose tested, 3000 ppm. The concentration of the test substance and mean weekly food intake values were used to determine the approximate doses received by the animals. 3000 ppm was approximately equal to a dose of 275 mg/kg bw/day in males and 310 mg/kg bw/day in females.

A supporting one-generation reproductive study (BioDynamics Inc., 1979b) is available, in which DTPMP-H was administered continuously via the diet to Long-Evans rats at concentrations of 300, 1000 and 3000 ppm through one complete generation. Test substance administration to the F0 generation females (20/group) was initiated at the onset of gestation and continued throughout the ensuing gestation and lactation periods. Administration then continued to the F1 generation animals (10 males and 20 females/group) through a growth period and mating, gestation and lactation period for two successive litters. The study was conducted according to a test protocol that is comparable to the appropriate OECD test guideline. It was not compliant with GLP. In the F0 generation, no treatment-related effects in the low and mid dose groups were evident. In the high dose group, females delivered litters containing fewer live pups and more dead pups (not statistically significant) resulting in a lower live birth index. Pups also had a lower weight at birth (not statistically significant). No other treatment-related effects were observed. In the F1 generation, no treatment-related effects were evident in the low dose group. No such effects were observed in the second litters and no other treatment-related effects were observed during the remainder of the study. Gross pathological examination of five adult F1 generation males and females of the control and high dose groups did not reveal any abnormal findings. A NOAEL of 3000 ppm, equivalent to 294 mg/kg bw/d for males and 312 mg/kg bw/d for females, was therefore concluded. The use of read-across data between members of the category is in accordance with the rationale outlined in the toxicological information endpoint summary of the IUCLID and Section 1.4 of the CSR.

The reduced premating exposure period in the read-across study with ATMP acid does not affect the validity of the study. It is reasonable to assume that in the available 3-generation reproductive toxicity study on ATMP the 60-day premating exposure would have been sufficient to allow a steady-state concentration of the test substance to be achieved based on the absorption, distribution and excretion profile of ATMP. Using a 60-day premating period is not a deficiency because only a small proportion of the dose is absorbed and subsequently distributed to all tissues/organs (except bone) and most of the absorbed dose is rapidly excreted in urine. Therefore, the full oestrus cycle will have been exposed to a steady state concentration of the test substance. This applies to ATMP and DTPMP as they have the same absorption, distribution and excretion profiles. In addition, no potential effect in reproductive organs has been demonstrated by DTPMP or ATMP. In a 90-day study in which sodium salt of DTPMP was administered to rats in their diets, no treatment-related adverse effects on the reproductive organs were observed (Williams, 1998). Also, in a 2-year chronic toxicity/carcinogenicity study (Biodynamics Inc., 1979d) in which ATMP was administered to rats in their diet there were no treatment-related adverse effects on reproductive organs.

The reproductive toxicity study on ATMP tested up to a dose (approximately 250-400 mg/kg bw/day; Biodynamics Inc., 1979a) that is a similar magnitude to the NOAEL for general systemic toxicity in the 2-year study for ATMP (500 mg/kg bw/day; Biodynamics Inc., 1979d). However, this 90-day NOAEL was based on no effects at the highest dose tested. Therefore, it is not surprising that toxicity was not observed in the parental animals in the 3-generation reproductive toxicity study on ATMP. By current standards the highest dose tested in both of these studies would have been higher; however, in this case, based on the extremely low oral absorption, limited distribution and rapid excretion data already described, increasing the administered dose 2-3 fold would still result in very low exposure to tissues, including all tissues relevant to reproductive toxicity, and therefore testing is not justifiable.


Effects on developmental toxicity

Description of key information

There is a good quality prenatal developmental toxicity screening study on DTPMP-xNa (Monsanto, 1982) in which mated female Sprague-Dawley rats (25/dose) were dosed by gavage at doses of 500, 1000, 2000 mg/kg bw/day (expressed as active acid). They were dosed on gestation days 6 to 19. Control animals were given 0.9% sodium chloride solution. On day 20 all surviving animals were sacrificed. There were no deaths prior to scheduled sacrifice. The highest dose produced marginal toxic effects in the dams, as evidenced by lower mean body weight gain between gestational days 6 and 20, and soft stools in some animals. No treatment-related lesions were detected at gross necropsy of the dams of any treatment group. There were no statistically significant treatment-related effects on postimplantation loss or fetal weight at any dose. Vertebral anomalies were observed in single fetuses in two litters of the 2000 mg/kg bw/day group and one of the 1000 mg/kg bw/day group. The incidence was not statistically significant, but the rare spontaneous occurrence of this type of malformation and the pattern of incidence was suggestive of a treatment-related effect. Review of these results for the REACH assessment concluded that the effects should not be considered adverse as they were not statistically significant and were only observed in the presence of maternal toxicity. Overall, the NOAEL for maternal toxicity was 1000 mg/kg bw/day, and for developmental toxicity was at least 2000 mg/kg bw/day (active acid).

A teratogenicity study (Biodynamics Inc., 1980; FDA segment II: teratological study; reliability score 2, comparable to OECD TG414 conducted prior to the adoption of OECD test guidelines and GLP, in which neutralised ATMP was administered by oral gavage to pregnant CD-1 mice (35 mated females/dose) on gestation days 6 -15. The doses tested were 100, 500 and 1000 mg/kg bw/day. The control group received the vehicle only. Parameters evaluated were mortality, body weight, clinical signs, uterine implantation data, ossification variation data and teratological evaluation. There were no treatment-related deaths (only deaths that occurred as a result of dosing errors and one death of a control animal on gestation day 11), no adverse clinical effects, no effects on body weights or body weight gains, and no effects on reproductive parameters (pregnancy rates, numbers of live and dead fetuses, implantations and resorptions). There were no treatment-related adverse findings during the macroscopic examination. No effects on mean foetal body weights, crown-rump length and foetal sex distribution. During the foetal skeletal evaluations, the incidence of foetuses with at least one ossification variation was slightly higher than the concurrent control in the mid and high dose groups. However, incidences for these groups were within the range of historical values for the laboratory and strain of mouse. The type and incidence of ossification variations during the skeletal evaluations were similar to the controls. However, there was a slight increase in the incidence of foetuses with rudimentary structures observed in the mid and high dose groups. The incidence of foetal external and soft tissue malformations was comparable between control and treated groups. No malformations were noted in the treated groups during the gross evisceration examinations. During the skeletal evaluations the incidence of malformations was low in the low (no malformations observed) and high dose groups. The incidence of skeletal malformations in the mid-dose group was significantly increased. However, this increase was attributed to a high number of malformed foetuses from a single mid-dose litter. Six foetuses from this litter had skeletal malformations that included misshapen tibia and fibula, angulated ribs and defective sternebrae. Since these effects were not observed in the highest and lowest dose groups they were not considered to be related to treatment. The NOAEL for maternal toxicity, foetal toxicity and teratogenicity was greater than 1000 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data. Treatment days were 09.04.1980 to 13.05.1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Guideline:
other: FDA "Guidelines for reproductive studies for evaluation of drugs for human use", segment II (teratological study)
Deviations:
not specified
Remarks:
No analytical evaluation of exposure levels.
Principles of method if other than guideline:
The study was designed to evaluate the embryotoxic and/or teratogenic potential of the test substance. Dosing was on gestation days 6-15, no measurement of gravid uterine weights and copora lutea were not counted.
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc.
- Age at study initiation: Females 57 days (males stated to be sexually mature)
- Weight at study initiation: Females on gestation Day 0 were approximately 26 g.
- Fasting period before study: No data
- Housing: Individual in elevated stainless steel cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: One month


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


IN-LIFE DATES: From: Day 6 of gestation: 09.04.1980 - 04.05.1980 To: Day 15 of gestation: 18.04.1980 - 13.05.1980.
Route of administration:
oral: gavage
Vehicle:
other: Not clear, stated to be distilled water and corn oil in different parts of the report.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of test substance were dissolved in distilled water and administered at a constant volume of 10 ml/kg bw/day. Dosing solutions were prepared fresh daily.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Overnight
- Verification of same strain and source of both sexes: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.
Duration of treatment / exposure:
GD 6 - 15
Frequency of treatment:
daily
Duration of test:
18 days
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
35 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes for mortality and gross signs of toxicological effects (no further details).
- Time schedule: Twice daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Gestation days 0, 6, 9, 12, 15 and 18.


BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 6, 9, 12, 15 and 18. Calculated body weight change for days 0-6, 6-15 and 15-18.


FOOD CONSUMPTION: No


WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18 (all surviving dams) and Day 18 post-mating in all surviving non-pregnant females.
- Organs examined: Complete gross pathology examination.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes, and weighed, measured and sex determined.
- Other: Live and dead fetuses. Internal sex determination of fetuses.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: No data
Statistics:
Maternal body weight and reproduction data: Bartlett's test followed by one-way ANOVA (equal variance) followed by Dunnett's test or Kruskal-Wallis test (unequal variance) and summed rank test (Dunn).  Pregnancy and fetal parameters: Chi square analysis followed by Fisher Exact test with Bonferroni correction. Armitage test for linear trend.
Indices:
No data
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no treatment-related deaths (only deaths that occurred as a result of dosing errors and one death of a control animal on gestation day 11), no adverse clinical effects, no effects on body weights or body weight gains, and no effects on reproductive parameters (pregnancy rates, numbers of live and dead fetuses, implantations and resorptions). There were no treatment-related adverse findings during the macroscopic examination.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects on mean fetal body w eights, crown-rump length and fetal sex distribution. During the fetal skeletal evaluations, the incidence of fetuses with at least one ossification variation was slightly higher than the concurrent control in the mid and high dose groups. However, incidences for these groups were within the range of historical values for the laboratory and strain of mouse. The type and incidence of ossification variations during the skeletal evaluations were similar to the controls. However there was a slight increase in the incidence of fetuses with rudimentary structures observed in the mid and high dose groups. The incidence of fetal external and soft tissue malformations were comparable between control and treated groups. No malformations were noted in the treated groups during the gross evisceration examinations. During the skeletal evaluations the incidence of malformations was low in the low (no malformations observed) and high dose groups. The incidence of skeletal malformations in the mid-dose group was significantly increased. However, this increase was attributed to a high number of malformed fetuses from a single mid-dose litter. Six fetuses from this litter had skeletal malformations that included misshapen tibia and fibula, angulated ribs and defective sternebrae. Since these effects were not observed in the highest and lowest dose groups they were not considered to be related to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1 Summary of Malformations found during the fetal skeletal examination.

 Group (mg/kg bw/day)  Malformation     Fetuses     Litter
     No. with malf./total examined  %  No. with malf fetuses/total examined  %
 0  None  0/138    0/24  
 100  Cervical vertebral defect 1/156   0.6  1/28  3.6
 500  Tibia misshapened - alone  1/203  0.5  1/34 2.9 
  - with misshapened fibula and angulated ribs  2/203   1.0  1/34  2.9
   - with misshapened fibula, angulated ribs and sternebrae defects   1/203  0.5  1/34  2.9
  - with angulated ribs  1/203  0.5  1/34  2.9
   Angulated ribs and scrambled sternebrae  1/203  0.5  1/34  2.9
   Cervical rib  1/203  0.5  1/34 2.9 
   5 lumbar vertebrae  2/203  1.0  1/34  2.9
   Total  9/203  4.4*  3/34  8.8
 1000  Scrambled sternebrae  1/183  0.5 1/32   3.1
   Vertebral defects  1/183  0.5  1/32  3.1

*Difference from the control group statistically significant p<0.05 (Fisher Exact test).

Conclusions:
In a well conducted teratogenicity study (FDA segment II: teratological study; reliability score 2) conducted prior to the adoption of OECD test guidelines and GLP, it was concluded that Dequest 2000 was not embryotoxic or teratogenic when administered to mice at 100, 500 or 1000 mg/kg bw/d by gavage on GD6-15.
Executive summary:

In a well conducted teratogenicity study (FDA segment II: teratological study; reliability score 2) conducted prior to the adoption of OECD test guidelines and GLP, Dequest 2000 was administered by oral gavage to pregnant CD-1 mice (35 mated females/dose) on gestation days 6 -15. The doses tested were 100, 500 and 1000 mg/kg bw/day. The control group received the vehicle only. Parameters evaluated were mortality, body weight, clinical signs, uterine implantation data, ossification variation data and teratological evaluation. There were no treatment-related deaths (only deaths that occurred as a result of dosing errors and one death of a control animal on gestation day 11), no adverse clinical effects, no effects on body weights or body weight gains, and no effects on reproductive parameters (pregnancy rates, numbers of live and dead fetuses, implantations and resorptions). There were no treatment-related adverse findings during the macroscopic examination. No effects on mean fetal body weights, crown-rump length and fetal sex distribution. During the fetal skeletal evaluations, the incidence of fetuses with at least one ossification variation was slightly higher than the concurrent control in the mid and high dose groups. However, incidences for these groups were within the range of historical values for the laboratory and strain of mouse. The type and incidence of ossification variations during the skeletal evaluations were similar to the controls. However there was a slight increase in the incidence of fetuses with rudimentary structures observed in the mid and high dose groups. The incidence of fetal external and soft tissue malformations were comparable between control and treated groups. No malformations were noted in the treated groups during the gross evisceration examinations. During the skeletal evaluations the incidence of malformations was low in the low (no malformations observed) and high dose groups. The incidence of skeletal malformations in the mid-dose group was significantly increased. However, this increase was attributed to a high number of malformed fetuses from a single mid-dose litter. Six fetuses from this litter had skeletal malformations that included misshapen tibia and fibula, angulated ribs and defective sternebrae. Since these effects were not observed in the highest and lowest dose groups they were not considered to be related to treatment. The NOAEL for maternal toxicity, fetal toxicity and teratogenicity was greater than 1000 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attachment for justification of read-across. ATMP acid is a member of the ATMP phosphonates category, structurally related to the DTPMP category.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across: supporting information
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Key result
Developmental effects observed:
no
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
DTPMP acid is a member of the DTPMP phosphonates category. The use of read-across data between members of the category is in accordance with the rationale outlined in the attached justification and Section 1.4 of the CSR.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across: supporting information
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.05.1980 to 09.06.1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The restrictions were that there was no analytical confirmation of dosing solutions, and there was no record of gravid uterine weight or the number of corpora lutea.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
No record of gravid uterine weight, number of corpora lutea not recorded, no analytical confirmation of dosing solutions.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: No data
- Weight at study initiation: 180-200 g
- Fasting period before study: No data
- Housing: Individually in suspended stainless steel mesh cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: None, animals were recieved mated and allocated to cages.


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ±2
- Humidity (%): 40-60
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 26.05.1980 To: 09.06.1980
Route of administration:
oral: gavage
Vehicle:
other: Aqueous solution
Details on exposure:
No data
Analytical verification of doses or concentrations:
no
Details on mating procedure:
No data. Animals were received on gestational day 1, having already been mated.
Duration of treatment / exposure:
GD 6 - 19
Frequency of treatment:
daily
Duration of test:
20 days
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
other: 0.9% (w/v) aqueous NaCl
Details on study design:
- Dose selection rationale: No data
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for visible toxic effects.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: Gestational days 3, 6, 8, 10, 13, 15, 17 and 20.


FOOD CONSUMPTION: No


WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Gross examination of all animals that included examination of external surfaces and thoracic and abdominal cavities.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
Comparison of body weights between treatment and control groups was performed using Dunnett's test. Counted data (corpora lutea, implants, resorptions, live and dead fetuses) and data expressed as percentage were analysed, when appropriate with the Mann-Whitney U test. Response data (pregnancy rates, number of litters with post-implantation loss, and fetuses or litters with abnormalities and variants) were analysed, when appropriate, with Fisher's exact test and the chi-square test.
Indices:
None
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No deaths occurred prior to the scheduled sacrifice. 9/25 dams had soft stools in the 2000 mg/kg bw/day group beginning on gestation day 14 (ninth day of treatment) and persisted through to gestation day 17. This finding was not observed in the other treated groups or the controls. The only statistically significant (P<0.01) effect observed was lower body weight gain (mean value approximately 68% of the control mean) between gestation day 6 and 20 for dams in the 2000 mg/kg bw/day group (33.6/27.6/27.5/22.9). However, terminal body weights were not statistically significantly affected (mean terminal body weights with uterine contents by dose: 360.1/368.1/357.0/346.1 and mean terminal bodyw eights without uterine contents: 265.8/264.5/260.1/257.2). There was no effect on pregnancy rate or the mean number of corpora lutea. There were no treatment-related findings in the gross necropsy.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no significant differences between any of the treatment groups and the control group in the number of pre- or postimplantation losses, resorptions or mean number of live fetuses. There were no effects on fetal body weight, sex ratios (males/litter: 5.6/5.8/5.9/4.8. Females/litter:  5.9/6.5/5.4/6.4). There were no treatment-related gross external malformations present. Subcutaneous haematomas were present in controls and all treated groups, however the incidence increased at 500 mg/kg bw/day (P<0.05) and was considered unrelated to treatment by the study authors (no dose relationship was present). The occurence of one female fetus that was hydrocephalic and one female with gastroschisis in the 1000 mg/kg bw/day group was considered by the study authors to be spontaneous. The hydrocephalic female also exhibited a developmental variation (underdeveloped renal papilla) that was not considered to be related to treatment.
Skeletal examination revealed single incidences of dwarfism (one female fetus of 500 mg/kg bw/day group) and fused sternebrae (one female fetus of the 2000 mg/kg bw/day group), which were considered spontaneous. Two fetuses from different litters in the 2000 mg/kg bw/day group and one fetus of the 1000 mg/kg bw/day group had vertebral anomalies (missing, reduced or fused vertebral arches). Although the incidence of these anomalies was not statistically significant compared with the control group, the rare spontaneous occurrence of such anomalies and the pattern of incidence indicated that they might have been treatment-related. Various skeletal variations occurred, but none showed a clear dose-response and so were considered spontaneous. Review of these results for the REACH assessment concluded that the effects should not be considered adverse as they were not statistically significant and were only observed in the presence of maternal toxicity.
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
In a good quality prenatal developmental toxicity screening study (reliability score 2) conducted using a protocol similar to OECD 414, and to GLP, clear maternal toxicity (approx. 30% decrease in body weight gain, soft stools) was noted in pregnant SD rats given 2000 mg/kg bw/day DTPMP (Dequest 2061; expressed as active acid) on GD 6-19 (NOAEL 1000 mg/kg bwt/d). The NOAEL for developmental toxicity was 2000 mg/kg bw/day (active acid).
Executive summary:

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

There were no statistically significant treatment-related effects on postimplantation loss or fetal weight at any dose. Vertebral anomalies were observed in single fetuses in two litters of the 2000 mg/kg bw/day group and one of the 1000 mg/kg bw/day group. The incidence was not statistically significant, but the rare spontaneous occurrence of this type of malformation and the pattern of incidence was suggestive of a treatment-related effect. Review of these results for the REACH assessment concluded that the effects should not be considered adverse as they were not statistically significant and were only observed in the presence of maternal toxicity.

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

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

There is a good quality prenatal developmental toxicity screening study on DTPMP-xNa (Monsanto, 1982) mated female Sprague-Dawley rats (25/dose) were dosed by gavage at doses of 500, 1000, 2000 mg/kg bw/day (expressed as active acid). They were dosed on gestation days 6 to 19. Control animals were given 0.9% sodium chloride solution. On day 20 all surviving animals were sacrificed. There were no deaths prior to scheduled sacrifice. The highest dose produced marginal toxic effects in the dams, as evidenced by lower mean body weight gain between gestational days 6 and 20, and soft stools in some animals. No treatment-related lesions were detected at gross necropsy of the dams of any treatment group. There were no statistically significant treatment-related effects on post-implantation loss or foetal weight at any dose. Vertebral anomalies were observed in single foetuses in two litters of the 2000 mg/kg bw/day group and one of the 1000 mg/kg bw/day group. The incidence was not statistically significant, but the rare spontaneous occurrence of this type of malformation and the pattern of incidence was suggestive of a treatment-related effect. Review of these results concluded that the effects should not be considered adverse as they were not statistically significant and were only observed in the presence of maternal toxicity. Overall, the NOAEL for maternal toxicity was 1000 mg/kg bw/day, and for developmental toxicity was at least 2000 mg/kg bw/day (active acid).

No further data on the developmental toxicity of DTMPM-xNa are available, however, information is available on DTPMP-H from a one-generation reproductive toxicity study (Biodynamics, 1979b), in which DTPMP-H was administered continuously via the diet to Long-Evans rats at concentrations of 300, 1000 and 3000 ppm through one complete generation. Test substance administration to the F0 generation females (20/group) was initiated at the onset of gestation and continued throughout the ensuing gestation and lactation periods. Administration then continued to the F1 generation animals (10 males and 20 females/group) through a growth period and mating, gestation and lactation period for two successive litters. The study was conducted according to a test protocol that is comparable to the appropriate OECD (415) test guideline. It was not compliant with GLP. In the F0 generation, no treatment-related effects in the low and mid dose groups were evident. In the high dose group, females delivered litters containing fewer live pups and more dead pups (not statistically significant) resulting in a lower live birth index. Pups also had a lower weight at birth (not statistically significant). No other treatment-related effects were observed. In the F1 generation, no treatment-related effects were evident in the low dose group. No such effects were observed in the second litters and no other treatment-related effects were observed during the remainder of the study. Gross pathological examination of five adult F1 generation males and females of the control and high dose groups did not reveal any abnormal findings. A NOAEL of 3000 ppm, equivalent to 294 mg/kg bw/d for males and 312 mg/kg bw/d for females, was therefore concluded. The use of read-across data between members of the category is in accordance with the rationale outlined in the Toxicological information endpoint summary of the IUCLID and Section 1.4 of the CSR.

No data are available on the developmental toxicity of any DTPMP category members to a second species, therefore good quality data from studies on rats and mice are read-across from ATMP-H.

ATMP acid has been tested in a pre-GLP teratogenicity study (Biodynamics Inc., 1979c; FDA segment II teratological study, comparable to OECD TG 414) in which neutralised ATMP acid was administered by oral gavage to pregnant Charles River CD rats (24/dose), at dose levels of 100, 500 and 1000 mg/kg bw/day, on gestation days 6 to 15. Control animals received the vehicle (water) only. Dams were sacrificed on gestation day 21 and recovered foetuses evaluated for external, soft-tissue and skeletal malformations. Maternal mortality, pregnancy rate, body weight gain, uterine implantation data, foetal size, sex data, ossification variation data and teratological evaluations were evaluated. High dose females gained less weight than the controls during the dosing period. A statistically significant increase in the number of resorptions was observed in the low and high dose animals (not the mid-dose). There was also an increase in the number of dams with two or more resorptions. However, the resorption data were within the range of historical values for the laboratory, so it was concluded that there was not a treatment-related effect. There were no teratogenic effects in the low and mid dose group. In the high dose group six foetuses from a single litter had common multiple malformations that included flexed forepaws, shortened and thickened torso, abdominal distension and exaggerated flexure of the head. Soft tissue examination revealed two of these foetuses had a malformation defect of the heart. The remaining high dose foetuses were generally unremarkable. Soft tissue and skeletal malformation data from the high dose group were similar to the control group. As the observations were only found in a single litter, born to a dam showing signs of toxicity (reduced body weight gain of 50%) it was concluded that the malformations were secondary to maternal toxicity. Therefore, the maternal NOAEL of ATMP was 500 mg/kg bw/day, and the NOAEL for foetotoxicity and teratogenicity was1000 mg/kg bw/day.

A teratogenicity study (Biodynamics Inc., 1980; FDA segment II: teratological study; reliability score 2, comparable to OECD TG414) conducted prior to the adoption of OECD test guidelines and GLP, in which neutralised ATMP was administered by oral gavage to pregnant CD-1 mice (35 mated females/dose) on gestation days 6 -15. The doses tested were 100, 500 and 1000 mg/kg bw/day. The control group received the vehicle only. Parameters evaluated were mortality, body weight, clinical signs, uterine implantation data, ossification variation data and teratological evaluation. There were no treatment-related deaths (only deaths that occurred as a result of dosing errors and one death of a control animal on gestation day 11), no adverse clinical effects, no effects on body weights or body weight gains, and no effects on reproductive parameters (pregnancy rates, numbers of live and dead fetuses, implantations and resorptions). There were no treatment-related adverse findings during the macroscopic examination. No effects on mean foetal body weights, crown-rump length and foetal sex distribution. During the foetal skeletal evaluations, the incidence of foetuses with at least one ossification variation was slightly higher than the concurrent control in the mid and high dose groups. However, incidences for these groups were within the range of historical values for the laboratory and strain of mouse. The type and incidence of ossification variations during the skeletal evaluations were similar to the controls. However, there was a slight increase in the incidence of foetuses with rudimentary structures observed in the mid and high dose groups. The incidence of foetal external and soft tissue malformations was comparable between control and treated groups. No malformations were noted in the treated groups during the gross evisceration examinations. During the skeletal evaluations the incidence of malformations was low in the low (no malformations observed) and high dose groups. The incidence of skeletal malformations in the mid-dose group was significantly increased. However, this increase was attributed to a high number of malformed foetuses from a single mid-dose litter. Six foetuses from this litter had skeletal malformations that included misshapen tibia and fibula, angulated ribs and defective sternebrae. Since these effects were not observed in the highest and lowest dose groups they were not considered to be related to treatment. The NOAEL for maternal toxicity, foetal toxicity and teratogenicity was greater than 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data for DTPMP acid and sodium salts and ATMP acid, no classification for reproductive and developmental toxicity is required for DTPMP (5-7Na) according to Regulation (EC) No 1272/2008.