Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reliable GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
Please refer to confidential details on test material.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Crl:WI(Han); Charles River Laboratories, Inc., Raleigh, NC
- Age at arrival: 62 days old
- Weight at study initiation: 168 g to 207 g (after arrival) and 171 g to 217 g (randomization and study assignment)
- Housing: individually housing except during cohabitation period
- Diet: Certified Rodent Diet, ad libitum
- Water: from individual bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25°C
- Humidity: 30 - 70%.
- Air changes: 10 changes per hour
- Photoperiod: 12-hours light: 12-hours dark

IN-LIFE DATES: From: 2012-07-01 To: 2012-07-20

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly, stored refrigerated (2°C to 8°C) and protected from light, and stirred continuously during sample collection and dose administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sample collection and analysis
Dose formulation samples were collected for analysis, stored refrigerated (2°C to 8°C) and protected from light until shipment for analysis. Formulation analyses were performed by HPLC.

Concentration analysis
Duplicate sets of samples (1 mL) were collected from the middle of the control and middle concentrations on the first day of preparation and all concentrations on the last day of preparation for concentration analysis and stored refrigerated (2°C to 8°C) and protected from light until shipment to the analytical laboratory. Duplicate sets of samples (1 mL) were collected from each specified concentration in the same manner on each occasion and were retained at the Testing Facility as backup samples.

Homogeneity analysis
Duplicate sets of samples (1 mL) were collected from the top, middle, and bottom of the low and high concentrations on the first day of preparation for homogeneity analysis and stored refrigerated (2°C to 8°C) and protected from light until shipment to the analytical laboratory. Duplicate sets of samples (1 mL) were collected from the low and high concentrations in the same manner on the same occasion and were retained at the Testing Facility as backup samples.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 5 days
- Proof of pregnancy: sperm in vaginal smear and/or a copulatory plug were considered to be DG0
- After successful mating each pregnant female was caged: individual
Duration of treatment / exposure:
Female rats were administered the test substance and/or the control substance formulations on DGs 6 through 20. Doses were adjusted based on the most recently recorded body weight and administered.
Frequency of treatment:
once daily at approximately the same time each day
Duration of test:
All female rats were euthanized on DG 21.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 250 and 700 mg/kg bw/day (20, 50 and 140 mg/mL) at a daily dose volume of 5 mL/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
25 rats per group
Control animals:
yes, concurrent vehicle
Details on study design:
Justification of route and dose levels:
The oral (gavage) route was selected for use because the exact dose can be accurately administered and to maximize systemic exposure. Dose levels were selected for this study on the basis of a range-finding maternal toxicity study in Wistar rats (further details see Section "Any other information on materials and methods".

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: twice during the acclimation period, on DG 0, daily before each dose was administered, and on the day of scheduled euthanasia.

POSTDOSE OBSERVATIONS
- Time schedule: 1 and 2 hours after dose administration and at the end of the normal working day

BODY WEIGHT
- Time schedule for examinations: twice during the acclimation period, on DG 0, daily during the dose and on the day of scheduled euthanasia.

FOOD CONSUMPTION
- Recorded: DGs 0, 6, 9, 12, 15, 18, and 21

MATING PERFORMANCE
- Mating performance was evaluated daily during the cohabitation period.

EUTHANASIA
Rats were euthanized by carbon dioxide asphyxiation. Live fetuses were euthanized by an intraperitoneal injection of sodium pentobarbital. On DG 21, female rats were Caesarean-sectioned, and examined for gross lesions. To minimize bias, Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dose group. In-life personnel replaced the complete cage tag with a white label bearing only the study number and the rat number prior to the time of Caesarean-sectioning and necropsy. Uteri of apparently nonpregnant animals were examined.

NECROPSY
A gross necropsy of the thoracic, abdominal, and pelvic viscera was performed for each rat.

TISSUE COLLECTION AND PRESERVATION
Representative samples of the tissues as indicated in Table 1 (please refer to "Any other information on material and methods incl. tables") were collected from all rats and were preserved in 10% neutral buffered formalin, unless otherwise indicated. A table of random units was used to select one control group rat from which all tissues examined at necropsy were retained, in order to provide control tissues for any possible histopathological evaluations of gross lesions.



Ovaries and uterine content:
OVARIAN AND UTERINE EXAMINATIONS
The ovaries and uterine content was examined after termination: Yes
-Gravid uterus weight: Yes
-Number of corpara lutea: Yes
- Number of implanations: Yes
- Number of early resorptions Yes
-Number of late resorptions: Yes

The reproductive tract was dissected from the abdominal cavity. The gravid uterus was excised and weighed. Corrected body weights (terminal body weight minus gravid uterine weight), as well as corrected body weight gains (corrected body weight minus body weight on DG 6), were calculated. The uterus was opened and the contents were examined. The fetuses were removed from the uterus and placed in individual containers. The ovaries and uterus were examined for number and distribution of corpora lutea, implantation sites, placentae (size, color or shape), live and dead fetuses, and early and late resorptions. Conception rate, pre-implantation loss and post-implantation loss (absolute and relative) were calculated. Uteri of apparently nonpregnant animals were examined while being pressed between glass plates to confirm the absence of implantation sites.
Fetal examinations:
External examinations: Yes [all per litter]
- Soft tissue and head examinations: Yes [half per litter]
- Skeletal examinations: Yes [half per litter]

Fetuses were examined for sex and for external abnormalities. Late resorptions were examined for external abnormalities and sex to the extent possible. The body weight of each fetus was recorded. Fetuses were individually identified with the study number, litter number, and uterine distribution.
Approximately one-half of the fetuses in each litter were examined for visceral abnormalities by using a modification of the microdissection technique of Staples. Each fetus was fixed in Bouin's solution, and the heads were subsequently examined by free-hand sectioning; head sections were stored in alcohol. The decapitated carcasses were not retained.
The remaining fetuses (approximately one-half of the fetuses in each litter) were examined for skeletal abnormalities after staining with alizarin red S. Malformations and variations were reported separately as well as combined. Following examination, skeletal preparations were retained in glycerin, with thymol added as a preservative.
Representative photographs of external, visceral, and skeletal abnormalities were taken. Abnormalities were classified as malformations and variations, including skeletal ossification parameters.
Statistics:
Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., maternal body weights, body weight changes, food consumption values and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p > 0.001)]. If the Analysis of Variance was significant (p ≤ 0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test is significant (p ≤ 0.001)], the Kruskal-Wallis Test was used (≤ 75% ties). In cases where the Kruskal-Wallis Test was statistically significant (p ≤ 0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyze the data. Count data (e.g., number of fetuses with a specified alteration) was evaluated using the procedures described above for the Kruskal-Wallis Test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
MORTALITY AND CLINICAL OBSERVATIONS
All rats survived to Day 21 of presumed gestation. Clinical signs attributed to administration of the test substance occurred at 250 and 700 mg/kg bw/day. Urine-stained abdominal fur occurred in each of the 4 dose groups, including the control group. However, the numbers of animals affected with the clinical sign was higher at 250 mg/kg bw/day and achieved statistical significance (p ≤ 0.01) at 700 mg/kg bw/day. This clinical sign was attributed to administration of the test substance. Mild dehydration (based on skin turgor) occurred in 1 animal at 250 mg/kg bw/day and in 3 animals at 700 mg/kg bw/day . Red perinasal substance occurred in 3 animals (significantly at p ≤ 0.01) at 700 mg/kg bw/day and was attributed to administration of the test substance. The remaining clinical signs at 700 mg/kg bw/day occurred in a single rat , primarily on the last day of dose administration or on the day of schedule euthanasia, and included the following: excess salivation (slight to moderate), thin body condition, urinestained perivaginal area, decreased motor activity, lost righting reflex, impaired righting reflex, low carriage, ptosis, piloerection, splayed hindlimbs, cold to touch, both ears pale, yellow fur around the mouth and perivaginal area, labored breathing, and bradypnea. All other clinical signs were considered unrelated to administration of the test substance.

MATERNAL BODY WEIGHTS AND BODY WEIGHT CHANGES AND GRAVID UTERINE WEIGHTS
Mean maternal body weight changes attributed to administration of the test substance, as well as changes that achieved statistical significance (p ≤ 0.01) relative to the respective control group values were noted at 700 mg/kg bw/day for the gestation period (uncorrected and corrected for gravid uterine weights).

On Days 6 to 9 and 18 to 21 of presumed gestation (DGs 6 to 9 and 18 to 21), significant reductions in mean body weight change (39% and 57% of the respective control group values) occurred at 700 mg/kg bw/day, and contributed to reductions in mean body weight change during the entire dose period (DGs 6 through 21; 76% of the control group value) and the overall gestation period (DGs 0 through 21; 81% of the control group value). Following correction for gravid uterine weights, mean body weight changes were also significantly reduced during the entire dose period (DGs 6 through 21C), and during the overall gestation period (DGs 0 through 21C) at 700 mg/kg bw/day (33% and 63% of the respective control group values).
Mean maternal body weights on DGs 20 and 21 were significantly reduced (p ≤ 0.05 and p ≤ 0.01; 95% and 92% of the respective control group values) at 700 mg/kg bw/day. Mean gravid uterine weights were generally comparable among the 4 dose groups and did not significantly differ (92%, 102%, and 93% of the control group value). Following correction for gravid uterine weights, mean maternal body weight on DG 21 (DG 21C) was significantly reduced (p ≤ 0.01; 92% of the control group value) at 700 mg/kg bw/day.

Mean maternal body weights and body weight gains (absolute and corrected for gravid uterine weights) were unaffected by administration of the test substance at dose levels as high as 250 mg/kg bw/day.

FOOD CONSUMPTION
Mean absolute and relative food consumption values at 700 mg/kg bw/day were significantly reduced (79% to 89% of the respective control group values) on DGs 6 to 9, 9 to 12, 12 to 15, and 18 to 21 within the dose period. As a result of these persistent reductions in food consumption values, corresponding reductions (86% to 92% of the respective control group values) in food consumption values occurred at 700 mg/kg bw/day during the entire dose period (DGs 6 through 21) and the overall gestation period (DGs 0 through 21). These reductions in food consumption values correlated with reductions in mean maternal body weight gains.

Mean absolute (g/day) and relative (g/kg bw/day) food consumption values were unaffected by administration of the test substance at dose levels as high as 250 mg/kg bw/day.

GROSS PATHOLOGY
There were no gross lesions attributed to administration of the test substance at dose levels as high as 700 mg/kg bw/day. A single rat in the 700 mg/kg bw/day group was observed with diaphragmatic hernia (a portion of the median lobe of the liver protruded into the thoracic cavity). This gross lesion was considered a congenital abnormality that was present prior to assignment to study and was not attributed to administration of the test substance. Persistent adverse clinical signs were confirmed at necropsy, when possible. These clinical signs included ungroomed coat, sparse hair coat, scab on the left forelimb, and red perinasal substance.

OVARIAN AND UTERINE EXAMINATION
No ovarian or uterine examination parameters were affected by dose levels of the test substance as high as 700 mg/kg bw/day.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
LITTER OBSERVATIONS
Pregnancy occurred in 23 to 25 rats in each dose group. Caesarean-sectioning observations were based on 23, 24, 24, and 25 pregnant rats with one or more live fetuses at 0 (control), 100, 250 and 700 mg/kg bw/day, respectively. Fetal body weight averages (total, male, and female) were significantly reduced (p ≤ 0.01; 93% to 94% of the respective control group values) at 700 mg/kg bw/day. These reductions in fetal body weight averages occurred at a maternally toxic dose level and were below the range of historical control data for the Testing Facility. The litter averages for implantations, percentage of preimplantation loss, litter sizes, live fetuses, early and late resorptions, percentage of postimplantation loss, percentage of resorbed conceptuses per litter, and percentage of live male fetuses per litter were comparable among the 4 dose groups and did not significantly differ. No dam had a litter consisting of only resorbed conceptuses, and there were no dead fetuses. All placentae appeared normal.
The mean number of corpora lutea at 100 mg/kg bw/day was statistically significantly lower (p ≤ 0.01), in comparison with the control group value. This lower mean number of corpora lutea was not attributed to administration of the test substance because implantation is essentially complete before DG 6, when dose administration was initiated and the mean litter size and the mean number of live fetuses per litter in this group, although lower in comparison with the respective control group values, were within the range of historical control data for the Testing Facility. In addition, the reduction did not occur in a dose-dependent manner.

FETAL ALTERATIONS
Fetal evaluations were based on 266, 241, 273, and 283 live DG 21, Caesarean-delivered fetuses in 23, 24, 24, and 25 litters at 0 (control), 100, 250 and 700 mg/kg bw/day, respectively. Of these respective fetuses, 128, 113, 130 and 140 fetuses were examined for soft tissue alterations, and 138, 128, 143 and 145 fetuses were examined for skeletal alterations and fetal ossification site averages.
There were 15 (65.2%), 13 (54.2%), 12 (50.0%) and 17 (68.0%) litters with fetuses with alterations in each of the 0 (control), 100, 250 and 700 mg/kg bw/day groups, respectively. The numbers of fetuses with any alteration observed were 23 (8.6%), 15 (6.2%), 22 (8.0%) and 31 (11.0%), and the percentages of fetuses with any alteration per litter were 8.8%, 6.9%, 7.8% and 13.3% in these same respective dose groups. Each of these parameters was generally comparable among the 4 dose groups and did not significantly differ.


MALFORMATIONS
There were no malformations attributed to administration of the test substance. Malformations occurred in the control group as well as the treated groups. A fetus in the control group had a short and constricted tail at fetal gross external examination. At skeletal examination, this fetus had 5 lumbar vertebrae present, no caudal vertebrae present, small arches in a sacral vertebra, and unilateral ossification in 2 sacral vertebrae. Another fetus in the control group had 12 thoracic vertebrae present, 5 lumbar vertebrae present, and 12 pairs of ribs present. One fetus at 700 mg/kg bw/day was observed with micrognathia, and the tongue was absent. Fusion of the right 14th thoracic vertebral centra to the left 1st lumbar vertebral centra and lumbar hemivertebra were present in another fetus in this dose group. These findings were only observed in single fetuses and were considered incidental and unrelated to administration of the test substance. The same applies to the parietal hole observed in 1 fetus in the 250 mg/kg bw/day group, which occurred in 1 fetus only, and without dose dependency.

VARIATIONS
External variations:
The right hindlimb was rotated medially in one fetus in the 700 mg/kg body weight/day dose group. No other alterations occurred in this fetus. No other fetal gross external alterations occurred on study.
Soft tissue variations:
No soft tissue variations occurred on study.
Skeletal variations:
Skull:
The frontal bones were incompletely ossified in 1 fetus in the 250 mg/kg body weight/day dose group. This fetus also had multiple thickened ribs. No other alterations occurred in this fetus.
Vertebrae:
One or both arches in the 7th cervical vertebrae had the appearance of arches in the 6th cervical vertebrae in 2 fetuses in the 250 mg/kg body weight/day dose group (no additional alterations). The left or right arch in the 6th cervical vertebra had the appearance of arches in the 7th cervical vertebra in 2, 1, 0, and 3 fetuses from 2, 1, 0, and 2 litters in the 0 (Control), 100, 250, and 700 mg/kg body weight/day dose groups, respectively. No additional alterations occurred in these fetuses. A bifid centrum in a thoracic vertebrae occurred in 1 and 2 fetuses from 1 and 2 litters in the 100 and 700 mg/kg body weight/day dose groups, respectively. Unilateral ossification of centra in 2 sacral vertebrae occurred in 1 fetus in the control group.
Ribs:
A cervical rib was present at the 7th cervical vertebra in 15, 5, 8, and 5 fetuses from 10, 5, 3, and 3 litters in the 0 (Control), 100, 250, and 700 mg/kg body weight/day dose groups, respectively. Both the litter and fetal incidences were significantly lower (p≤ 0.01) in the 100, 250, and
700 mg/kg body weight/day dose groups, in comparison with the respective control group values.
Wavy ribs, a reversible delay in ossification occurred in 3, 9, 9, and 14 fetuses from 2, 9, 7, and 8 litters in the 0 (Control), 100, 250, and 700 mg/kg body weight/day dose groups, respectively. The majority of the remaining fetuses also had thickened ribs. One or more thickened ribs occurred in 3, 7, 12, and 12 fetuses in 2, 7, 9, and 9 litters in the 0 (Control), 100, 250, and 700 mg/kg body weight/day dose groups, respectively.
Fetal ossification:
The mean number of ossified caudal vertebrae and the mean number of ossified hindlimb tarsals, metatarsals, and phalanges were reduced or significantly reduced (p≤ 0.05) in the 700 mg/kg body weight/day dose group, in comparison with the respective control group values. The mean
number of ossified hindlimb tarsals was slightly lower (80% of the control group value) in the 250 mg/kg body weight/day dose group but did not achieve statistical significance. No other biologically important or statistically significant differences occurred among the dose groups in the average numbers of ossification sites per fetus for the hyoid, vertebrae (cervical, thoracic, lumbar, and sacral), ribs, sternum (manubrium, sternal centers, and xiphoid), or forelimbs (carpals, metacarpals, and phalanges).

No other skeletal variations occurred on study.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Mean body weight gain [g] in pregnant rats treated by oral gavage with the test item from days of gestation 6 through 20 (DGs 6‑20)

Study period

Dose level [mg/kg bw/day]

0

100

250

700

Female rats

 

Gestation [DG 0-21]

128.9

124.6

134.8

104.4**

Gestation [DG 0-21]corr.

51.5

53.7

56.2

32.7**

corr.: corrected for gravid uterine weights, DG: day of gestation

** significantly different from the control group value (p ≤ 0.01), Dunnett’s test

Table 2: Mean fetal body weights [g] at Caesarean-sectioning on DG 21

Group

Dose level [mg/kg bw/day]

0

100

250

700

Male fetuses

5.12

5.31

5.14

4.75**

Female fetuses

4.84

5.02

4.90

4.53**

Male and female fetuses

4.96

5.13

5.05

4.63**

DG: Day of gestation

** significantly different from the control group value (p ≤ 0.01), Dunnett’s test; value below lower limit of historical control data range

Table 3: Ossification sites per fetus per litter (mean ± SD)

Site

Dose level [mg/kg bw/day]

0

100

250

700

Vertebrae

 

Cervical

7.00 ± 0.00

7.00 ± 0.00

7.00 ± 0.00

7.00 ± 0.00

Thoracic

13.40 ± 0.32

13.50 ± 0.25

13.56 ± 0.33

13.44 ± 0.22

Lumbar

5.58 ± 0.32

5.50 ± 0.25

5.43 ± 0.33

5.57 ± 0.23

Sacral

3.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

3.00 ± 0.00

Caudal

7.17 ± 0.92

7.41 ± 0.76

7.37 ± 0.48

6.65 ± 0.63*

Hindlimba

 

Tarsals

0.40 ± 0.35

0.51 ± 0.34

0.32 ± 0.30

0.22 ± 0.26

Metatarsals

4.99 ± 0.04

4.99 ± 0.04

4.98 ± 0.06

4.92 ± 0.14*

Digits

5.00 ± 0.00

5.00 ± 0.00

5.00 ± 0.00

5.00 ± 0.00

Phalanges

7.47 ± 1.06

7.60 ± 1.12

7.32 ± 1.08

6.70 ± 1.13*

SD: standard deviation; the number of litters examined per group ranged from 23 to 25, the number of fetuses examined ranged from 128 to 145.

a:  calculated as average per limb

*   significantly different from the control group value (p ≤ 0.05), Dunnett’s test

Dose Formulation Analyses

Analysis of the dose formulation samples revealed all actual concentrations were within the acceptance criteria of ± 15% of the respective theoretical concentrations. All dose formulation samples met acceptance criteria for homogeneity (the relative standard deviation [RSD] of concentrations was < 5% for each group). Control substance samples contained no detectable concentrations of the test substance.

Data from maternal toxicity range-finding study in Wistar Han Rats (BASF 2013; 10R0473/11X485)

Urine-stained abdominal fur, slight to moderate excess salivation and ungroomed coat occurred in a generally dose-dependent manner in each of the 100, 300, and 1000 mg/kg body weight/day dose groups and were attributed to administration of the test substance. Additional clinical signs including dehydration (based on skin turgor), piloerection, discolored (bright yellow or yellow) urine, soft or liquid feces, hunched posture, scant feces, decreased motor activity, and discolored fur occurred in the 300 and/or 1000 mg/kg body weight/day dose groups and were also attributed to administration of the test substance. Ptosis, thin body condition, and hyperpnea occurred in 1 rat in the 1000 mg/kg body weight/day dose group. Mean maternal body weights and body weight changes (absolute and corrected for gravid uterine weights), food consumption values (absolute and relative), gravid uterine weights, and terminal body weights were reduced and absolute and relative liver weights were increased in the 1000 mg/kg body weight/day dose group.

No mortalities or gross lesions attributed to administration of C-1701 B_C_3 occurred during study, and mean maternal body weights and body weight changes (absolute and corrected for gravid uterine weights), food consumption values (absolute and relative), gravid uterine weights, terminal body weights, and liver weights (absolute and relative) were unaffected by administration of the test substance at dose levels as high as 300 mg/kg body weight/day.

Based on these data, dose-level selections for the definitive prenatal developmental toxicity study in Wistar rats, as well as the reproduction/developmental toxicity screening test in Wistar rats, were 0 (Control), 100, 250, and 700 mg/kg body weight/day; the highest dose level was expected to produce maternal toxicity.

Applicant's summary and conclusion