Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
29.10.1976 to 15.08.1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well documented and meets generally accepted scientific principles, but was not conducted in compliance with GLP. The study is read across from ATMP (CAS 6419-19-8).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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 wks
- 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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow (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 (how): 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.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 300, 1000 or 3000 ppm
Basis:
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

Examinations

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.
Estrous 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 behavioural 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.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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".  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. The was no dose response and the deaths were considered sporadic, therefore not related to treatement.
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): 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.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Dose descriptor:
other: NOAEL F3
Effect level:
3 000 ppm
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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): 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.

OTHER LITTER PARAMETERS: Sex ratio was not affected by treatment.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
3 000 ppm

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
3 000 ppm

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables


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

         Males (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 (%)        % 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 (%)        % 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 (%)        % 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.




Applicant's summary and conclusion

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 axamination 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.