Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The toxicity to reproduction assessment of SPE1415 is based on read-across data from the structural analogues ATMP (acid) and DTPMP (acid).

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 (acid) 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). In a well conducted and documented, pre-GLP one generation reproductive toxicity study DTPMP (acid) administered via the diet caused no clear treatment-related or statistically significant effects in rats at doses up to 3000 ppm in diet. A NOAEL of 3000 ppm (equivalent to 294 mg/kg bw/d for males and 312 mg/kg bw/d in females) was therefore concluded.



Link to relevant study records
Reference
Endpoint:
three-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.
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 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
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.
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
300 ppm (nominal)
Remarks:
See table 3 for conversion to mg/kg bw/day
Dose / conc.:
1 000 ppm (nominal)
Remarks:
See table 3 for conversion to mg/kg bw/day
Dose / conc.:
3 000 ppm (nominal)
Remarks:
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 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.
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: 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".  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.
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
other: Nominal in diet
Sex:
male/female
Basis for effect level:
other: General and reproductive toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
other: Nominal in diet
Sex:
male/female
Basis for effect level:
other: General and reproductive toxicity
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): 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.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
3 000 ppm
Based on:
other: Nominal in diet
Sex:
male/female
Basis for effect level:
other: General and reproductive toxicity
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
3 000 ppm
Based on:
other: Nominal in diet
Sex:
male/female
Basis for effect level:
other: General and reproductive toxicity
Key result
Dose descriptor:
NOAEL
Generation:
other: F3
Effect level:
3 000 ppm (nominal)
Based on:
other: Nominal in diet
Sex:
male/female
Basis for effect level:
other: General and reproductive toxicity
Reproductive effects observed:
not specified


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.




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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
275 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Exceeds requirements
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

The developmental toxicity assessment of SPE1415 is based on read-across data from the structural analogues ATMP (acid) and DTPMP (sodium salt). Additional supporting data on another confidential analogue substance is discussed in Additional Information.

In a well documented pre-GLP teratology study, ATMP (acid) was not embryotoxic or teratogenic when administered to rats at 100 or 500 mg/kg bw/d by gavage on GD6-15. At 1000 mg/kg bw/d, six fetuses from a single litter showed common multiple malformations in presence of a 50% decrease in individual maternal body weight gain (possibly indicative of concurrent maternal toxicity); all other high dose fetuses were normal. The clear absence of any comparable effect in other high dose litters and lack of dose-response indicates that 1000 mg/kg bwt/d was a probable no-effect level for embryotoxicity and fetotoxicity. The maternal NOAEL was 500 mg/kg bw/day. In a good quality developmental toxicity screening study 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 (sodium salt) on GD 6-19 (NOAEL 1000 mg/kg bwt/d). The NOAEL for developmental toxicity was 2000 mg/kg bw/day (as active acid).


Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
No data. Dates of treatment were 27.12.1978 to 19.01.1979
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.
Guideline:
other: FDA "Guidelines for reproductive studies for evaluation of drugs for human use", segment II (teratological study)
Deviations:
not specified
Remarks:
Treatment on GD 6 - 15; no record of gravid uterine weight; number corpora lutea not recorded; no analytical confirmation of exposure levels.
Principles of method if other than guideline:
Study was used to assess the teratogenic and/or embryotoxic potential of the test substance.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: gavage
Vehicle:
water
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
- Further matings after two unsuccessful attempts: No data
- 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:
16 days
No. of animals per sex per dose:
24 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: 21 d
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

MATERNAL TOXIC EFFECTS BY DOSE LEVEL AND BY SEX
Effects with dose level: 0/100/500/1000 mg/kg bw/d

Mortality
- one female from 100 mg/kg bw/d group moribund and sacrificed on GD6 (first day of treatment)

Body weight
- no significant differences in maternal bwt gain between groups
- body weight gain GD6-15: 50/49/50/44
- 12% (non-significant) reduction in body weight gain at 1000 mg/kg bw/d on GD6-15
Comment: individual body weight gain for dam 822 (high dose) on GD6-15 = 22 g; mean gain for controls = 50 g; mean gain for high dose group = 44 g. Bwt gain for this dam on GD 0-6 (preceding treatment) and on GD 15-21 (post-treatment) was similar or greater than mean bwt gain for control and high dose group.

Clinical observations, physical signs
- none present

Necropsy findings
- few adverse changes present, no treatment related effects 

Reproductive parameters
- pregnancy rate comparable between groups (100% in control, mid and high dose groups, 95.6% at 100 mg/kg bw/d), no
treatment-related changes.
- mean number of corpora lutea (17.2/16.0/16.7/16.8), implantations (14.5/14.7/14.0/13.7) and implantation efficiency (84.3%/91.8%/84.0%/81.2%) comparable; significant 7% decrease in corpora lutea and significant 7.5% increase
in implantation efficiency at 100 mg/kg bwt/d (P<0.05 in both instances) considered unrelated to treatment by authors
- mean number live fetuses comparable: 14.3/13.9/13.5/12.9 (no dead fetuses in any group)
- mean number resorptions: 0.2/0.8(P<0.05)/0.5/0.8(P<0.05); all values within historical control range
- non-dose related, non-significant increase in dams with 2 or more resorptions in treated groups (0%/22.7%/8.3%/20.8%);
within historical control range

FETAL DATA
Effects with dose level: 0/100/500/1000 mg/kg bw/d
- body weight by sex: males 5.54/5.43/5.71/5.49; females 5.25/5.17/5.34/5.16 (no significant effect)

Crown-rump length: males 4.2/4.2/4.3 (P<0.01)/4.2; females 4.1/4.1/4.2 (P<0.01)/4.1 (increase at 500 mg/kg bw/d
considered biologically insignificant by authors)

Sex: males/litter 6.9/7.0/6.6/6.0; females 7.4/6.9/7.0/6.9 (no significant effect)

Sex ratio (m:f): 92.4%/102.0%/94.6%/87.3% (no significant effect)

Variation in ossification: fetuses 80.2%/83.5%/79.3%/84.3%; litters 95.8%/100.0%/100.0%/100.0% (no significant effect)

External malformations: 
- incidence: 0 fetuses from 339 examined/0 from 305 examined/0 from 325 examined/6 from 315 examined
-  in the high dose group, one female (no. 822) had 6 fetuses (from a total litter of 16) with a syndrome of
defects that included: flexed forepaws, shortened and thickened torso, abdominal distension and exaggerated
forward flexure of the head (see maternal body weight, above). Remaining high dose fetuses (n = 309) unremarkable.

Total skeletal malformations 
- total fetuses examined: 177/158/169/159
- per fetus: 4.0%/1.9%/3.0%/1.3% (no significant effect)
- per litter: 20.8%/13.6%/20.8%/8.7% (no significant effect)
- type of skeletal malformations control: angulated ribs, cervical rib, wavy rib
100 mg/kg: angulated rib, cervical rib, angulated and wavy rib
500 mg/kg: cervical rib, angulated and wavy rib, 7 lumbar vertebra
1000 mg/kg: 5 lumbar vertebra, fused sternebrae 

Total soft tissue malformations
- total fetuses examined: 162/147/156/150
- per fetus: 4.3%/8.2%/4.5%/4.0% (no significant effect)
- per litter: 16.7%/40.9%/29.2%/20.8% (no significant effect)
- type of soft tissue malformation
control: distended renal pelvis, renal pelvis, ureter, baldder
100 mg/kg: as control + fold in retina
500 mg/kg: as control + ectopic kidney
1000 mg/kg: as control + fold in retina, anophthalmia, malrotation of heart
- malrotation of the heart occurred in high dose 2 fetuses, both from litter No. 822 (see "maternal body weight"  and
"external malformations", above)

Visceral malformations
- per fetus: 0.6%/1.3%/0.6%/1.9% (no significant effect)
- per litter: 4.2%/9.1%/4.2%/12.% (no significant effect)
- type of visceral malformation control: distended ureter
100 mg/kg: distended ureter +/- renal pelvis 500 mg/kg: as control
1000 mg/kg: as control + malpositioned testis

Conclusions:
In a well documented pre-GLP teratology study (FDA segment II teratological study; reliability score 2) Dequest 2000 was not embryotoxic or teratogenic when administered to rats at 100 or 500 mg/kg bw/d by gavage on GD6-15. At 1000 mg/kg bw/d, six fetuses from a single litter showed common multiple malformations in presence of a 50% decrease in individual maternal body weight gain (possibly indicative of concurrent maternal toxicity); all other high dose fetuses were normal. The clear absence of any comparable effect in other high dose litters and lack of dose-response indicates that 1000 mg/kg bwt/d was a probable no-effect level for embryotoxicity and fetotoxicity. The maternal NOAEL was 500 mg/kg bw/day.
Executive summary:

In a well documented pre-GLP teratology study (FDA segment II teratological study; reliability score 2) Dequest 2000 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 to15. Control animals received the vehicle (water) only. Dams were sacrificed on gestation day 21 and recovered fetuses evaluated for external, soft-tissue and skeletal malformations. Maternal mortality, pregnancy rate, body weight gain, uterine implantation data, fetal 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 fetuses from a single litter had common multiple malformations that included flexed forepaws, shortened and thickened torso, abdominal distention and exaggerated flexure of the head. Soft tissue examination revealed two of these fetuses had a malformation defect of the heart. The remaining high dose fetuses were generally unremarkable. Soft tissue and skeletal malformation data from the high dose group were similar to the control group. Although a possible teratogenic effect could not be excluded, it was most likely that the effects were secondary to maternal toxicity. Therefore the maternal NOAEL was 500 mg/kg bw/day, and the NOAEL for fetotoxicity and teratogenicity was >1000 mg/kg bw/day.

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
Quality of whole database:
Exceeds requirements
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Reliable studies on reproductive and developmental toxicity conducted on structural analogues of the substance all gave negative results. Therefore there is no justification for classification of the substance.