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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 02, 2009 to July 08, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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:
liquid

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)1
Details on test animals and environmental conditions:
SOURCE

Sexually mature, virgin female Crl:CD(SD) rats from Charles River Laboratories, Inc., Raleigh, NC, were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. Test facility has historical control data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat.

ANIMAL RECEIPT AND ACCLIMATION

The animals were approximately 70 days old upon receipt. Each female was examined by a qualified technician on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number and housed for 14 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and general changes in appearance and behavior. Body weights were recorded prior to the initiation of breeding.

ANIMAL HOUSING

Upon arrival and until pairing, all rats were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. The cage-board was changed at least three times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages; nesting material was not required, as the females were euthanized prior to the date of expected parturition. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The animal facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

DIET, DRINKING WATER, AND MAINTENANCE

The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, is a certified feed with appropriate analyses performed by the manufacturer. Feed lots used during the study are documented in the study records. Feeders were changed and sanitized once per week. Municipal water supplying the facility is regularly sampled for contaminants. The results of the diet and water analyses are maintained at the test facility.

ENVIRONMENTAL CONDITIONS

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20% relative humidity. Room temperature and relative humidity were controlled and monitored using the Metasys® DDC Electronic Environmental control system. Actual mean daily temperature ranged from 70.2°F to 71.2°F (21.2°C to 21.8°C) and mean daily relative humidity ranged from 43.6% to 52.7% during the study. Light timers were calibrated to provide a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan), once daily during gestation days 6-19. The dose volume for all groups was 10 mL/kg. Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance formulations were weight/weight (test substance/vehicle) mixtures. The test substance formulations were prepared approximately weekly as single formulations for each dose level, divided into aliquots for daily dispensation, and stored at room temperature.
Prior to the initiation of dose administration, samples for homogeneity determination, resuspension homogeneity, and stability and concentration analysis were collected. All analyses were conducted using a validated high performance liquid chromatography method using ultraviolet absorbance detection.
The analyzed dosing formulations were within the standard operating procedures range for suspensions (85% to 115%), were homogeneous, and were stable after 10 days of room temperature storage.
Details on mating procedure:
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding.
Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was prepared. The selected females were approximately 12 weeks old when paired for breeding. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.


Duration of treatment / exposure:
from gestation days (GD) 6 through 19
Frequency of treatment:
once daily
Duration of test:
90 d
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
8
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance, in the vehicle, deionized water, was administered orally by gavage to five groups of eight bred female Crl:CD(SD)1 rats once daily from gestation days (GD) 6 through 19. Dose levels were 15, 50, 150, 500, and 1000 mg/kg/d administered at a dose volume of 10 mL/kg. A concurrent control group composed of eight bred females received the vehicle on a comparable regimen.

The bred females were assigned to groups using a WTDMS computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Replacement animals were arbitrarily assigned based on body weight. One female in the 1000 mg/kg/d group was found dead on gestation day 7. The cause of death for this female was determined to be an intubation error. Subsequently, this female was replaced with another female. Body weight values ranged from 233 g to 284 g on gestation day 0.

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS AND SURVIVAL

All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS

Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20. When body weights could not be determined for an animal during a given interval (due to an unscheduled death), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables. Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0 to 20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION

Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/d and g/kg/d for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, food spillage, obvious erroneous value, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables.
Ovaries and uterine content:
ANATOMIC PATHOLOGY AND LAPAROHYSTERECTOMY UNSCHEDULED DEATHS

A gross necropsy was performed on females that died or were euthanized (by carbon dioxide inhalation) in extremis during the course of the study. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The number and location of implantation sites, corpora lutea, and viable fetuses were recorded. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). The maternal animals and all products of conception were then discarded.

GESTATION DAY 20 LAPAROHYSTERECTOMY

The laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving rats were euthanized on gestation day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the post mortem findings were correlated with the ante mortem comments, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The carcass of each female was then discarded. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
Fetal examinations:
Fetal examinations were performed blind to treatment group. A detailed external examination of each fetus was conducted to include, but was not limited to, an examination of the eyes, palate, and external orifices. Findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life). Each fetus was weighed, sexed, euthanized by hypothermia followed by an intrathoracic injection of sodium pentobarbital (if necessary), and discarded.
Statistics:
All statistical tests were performed using the WTDMS™ Management System. Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Data obtained from nongravid animals were excluded from statistical analyses.
Statistical analyses were not conducted if the number of animals was two or less. Due to the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ by ±1 in the last significant figure. Where applicable, the litter was used as the experimental unit.
Mean maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings noted for the females in the 1000 mg/kg/d group on the day prior to or day of death or euthanasia included prostration, hypoactivity, clonic convulsions, gasping, rales, shallow respiration, red and/or clear material around the nose and mouth, and dilated pupils; these findings were noted at the daily examinations or 1 hour following dose administration. Additionally, two females in the 500 mg/kg/d group were euthanized in extremis on gestation day 9 or 14. These females in the 500 mg/kg/d group were noted with increased respiration, decreased defecation, rales, gasping, labored respiration, and/or red and/or clear material around the nose, mouth, and/or forelimbs on the day of or day prior to euthanasia. All other animals survived to the scheduled necropsy.
Clinical findings observed for surviving females in the 500 mg/kg/d group at the daily examinations or approximately 1 hour following dose administration included rales, labored respiration, hair loss on the hindlimbs and/or thoracic and/or abdominal areas, clear material around the nose, mouth, eye and/or forelimbs, salivation, and/or dilated pupils.
Clinical findings noted in the 15, 50, and 150 mg/kg/d groups at the daily examinations or 1 hour following dose administration, including hair loss on various body surfaces, red and/or clear material around the mouth and/or nose, and/or yellow material on the urogenital area, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
All females in the 1000 mg/kg/d group were found dead or euthanized in extremis on gestation day 6, 7, or 8 (following 1-3 doses). Most females had body weight losses and reduced food consumption prior to death or euthanasia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significant (p<0.01) mean body weight loss was noted in the 1000 mg/kg/d group at the onset of treatment (gestation day 6-7). Additionally, a body weight loss was noted for the one remaining female in the group on gestation day 7-8. Further evaluation of body weight data was precluded by death or euthanasia of all females in this group by gestation day 8.
In the 500 mg/kg/d group, a significant (p<0.05) mean body weight loss was noted during gestation days 6-9. Mean body weight gains in this group were significantly (p<0.01) lower during gestation days 15-20. When the overall treatment period (gestation day 6-20) was evaluated, mean body weight gain in the 500 mg/kg/d group was significantly (p<0.01) lower than the control group. The mean body weight in the 500 mg/kg/d group was significantly (p<0.05) lower (12.8%) than the control group on gestation day 20. Additionally, mean net body weight and net body weight gain in the 500 mg/kg/d group were lower (significant at p<0.01 for net body weight gain) than the control group. Mean gravid uterine weight in the 500 mg/kg/d group was lower than the control group primarily due to a single female with one implantation.
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 15, 50, and 150 mg/kg/d groups were similar to that in the control group. The slight differences were not statistically significant and did not occur in a dose-related manner.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly (p<0.01) lower mean food consumption was noted in the 1000 mg/kg/d group at the onset of treatment (gestation day 6-7). Additionally, lower food consumption (6 g/animal/d) was noted for the one remaining female in the group on gestation day 7-8. Further evaluation of food consumption data in the 1000 mg/kg/d group was precluded by death or euthanasia of all females in this group by gestation day 8.
In the 500 mg/kg/d group, significantly (p<0.01 or p<0.05) lower mean food consumption was noted throughout the gestation treatment period (gestation days 6-9, 9-12, 12-15, 15-20, and 6-20). Lower mean food consumption noted in the 500 mg/kg/d group correlated to lower mean body weight gains during the treatment period.
Mean food consumption in the 15, 50, and 150 mg/kg/d groups was similar to that in the control group. The slight differences in food consumption were not statistically significant and did not occur in a dose-related manner.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All eight females in the 1000 mg/kg/d group were found dead or euthanized in extremis by gestation day 8. Additionally, two females in the 500 mg/kg/d group were euthanized in extremis on gestation day 9 or 14. All other females survived to the scheduled necropsy on gestation day 20. Macroscopic findings in both the 500 and 1000 mg/kg/d groups consisted of distended stomach and red material on the mouth and nasal area. In the 500 mg/kg/d group, one female had a distended stomach, duodenum, jejunum, ileum, cecum, and colon, and another female had thick vaginal contents. All females were gravid with the exception of one female each in the 50 and 1000 mg/kg/d groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
A decrease in the mean number of viable fetuses and an increase in the mean litter proportion of pre-implantation loss was noted at 15 (12.9 per dam and 15.0% per litter, respectively) and 500 mg/kg/d (12.2 per dam and 21.5% per litter, respectively) compared to the concurrent control group (15.1 per dam and 7.1% per litter, respectively). This was due to one female in the 15 and 500 mg/kg/d groups that had one to three implantations. Because implantation occurs prior to the initiation of test substance administration, these differences were not attributed to test substance administration. Mean numbers of corpora lutea and implantation sites were similar across all groups. The slight differences from the control group were not statistically significant.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Evaluation of laparohysterectomy parameters in the 1000 mg/kg/d group was precluded by mortality and moribundity. Intrauterine growth and survival were unaffected by test substance administration at dose levels of 15, 50, 150, and 500 mg/kg/d.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
The test substance-related toxicity was evidenced by reductions in mean maternal body weights, body weight gains, and food consumption at dose levels of 500 and 1000 mg/kg/d when test substance was administered orally by gavage to bred Crl:CD(SD) rats. In addition, the maximum tolerated dose was exceeded at 500 and 1000 mg/kg/d as evidenced by mortality and moribundity. There was no evidence of maternal toxicity at 15, 50, and 150 mg/kg/d.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Effect levels (fetuses)

Remarks on result:
not measured/tested

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the maternal and developmental NOAEL of the test substance were 150 and 500 mg/kg bw/day, respectively.
Executive summary:

A study was conducted to determine dose levels of the test substance for a possible definitive prenatal developmental toxicity study in rats according to OECD Guideline 414 and EPA OPPTS 870.3700, in compliance with GLP. The test substance in the vehicle, deionized water, was administered orally by gavage to 5 groups of 8 bred female Crl:CD(SD)1 rats once daily from Gestation Days (GD) 6 through 19. Dose levels were 15, 50, 150, 500 and 1000 mg/kg bw/day administered at a dose volume of 10 mL/kg bw. A concurrent control group composed of 8 bred females received the vehicle on a comparable regimen. The females were approximately 13 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity, and individual clinical observations were recorded from GD 0 to 20. Body weights and food consumption were recorded on GD 0 and 6-20 (daily). On GD 20, a laparohysterectomy was performed on each surviving female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external malformations and developmental variations. All eight females in the 1000 mg/kg bw/day group were found dead or euthanized in extremis on GD 6, 7 or 8. These females were noted with rales, hypoactivity, prostration, clonic convulsions, gasping, shallow respiration, red and/or clear material around nose and/or mouth, and dilated pupils at the daily examination or approximately 1 h following dose administration 1 d prior to or on the day of death or euthanasia. Additionally, body weight losses and reduced food consumption prior to death or euthanasia were noted for these females. The mortality at 1000 mg/kg bw/day precluded the evaluation of laprohysterectomy parameters and fetal morphology at this dose level. Two females in the 500 mg/kg bw/day group were euthanized in extremis on GD 9 or 14. Females in this group, including the two females that were euthanized in extremis, were noted with rales, increased respiration, dilated pupils, gasping, decreased defecation, labored respiration, salivation and/or clear and/or red material around the nose, mouth and/or forelimbs at the daily examinations or approximately 1 h following dose administration. All other females in the control, 15, 50 and 150 mg/kg bw/day groups survived to the scheduled necropsy on GD 20. There were no test-substance clinical findings noted at 15, 50 and 150 mg/kg bw/day at the daily examinations or approximately 1 h following dose administration. Lower mean body weight gain and decreased food consumption were noted at 500 mg/kg bw/day throughout the entire treatment period GD 6-20. As a result of lower mean body weight gains during gestation, mean body weights, net body weight gain and net body weight in this group were lower than the controls. A slightly lower mean gravid uterine weight was noted in the 500 mg/kg bw/day group due to a single female with one implantation. Mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 15, 50 and 150 mg/kg bw/day groups were similar to controls. At necropsy, two and one femalesat 500 and 1000 mg/kg bw/day, respectively, were noted with a distended stomach, and/or gas-filled distended stomach, duodenum, jejunum, ileum, cecum and colon. One female in the 500 mg/kg bw/day group (euthanized in extremis on GD 14) was noted with thick brown contents in the vagina. There were no internal findings noted at 15, 50 and 150 mg/kg bw/day. Intrauterine growth and survival at 15, 50, 150 and 500 mg/kg bw/day were similar to controls. There were no external malformations or developmental variations observed in this study. In conclusion, test substance-related toxicity was evidenced by reductions in mean maternal body weights, body weight gain and food consumption at dose levels of 500 and 1000 mg/kg bw/day when test substance was administered orally by gavage to bred Crl:CD(SD) rats. In addition, the maximum tolerated dose was exceeded at 500 and 1000 mg/kg bw/day as evidenced by mortality and moribundity. There was no evidence of maternal toxicity at 15, 50, and 150 mg/kg bw/day. Intrauterine growth and survival and fetal morphology were unaffected by maternal test substance administration at 15, 50, 150 and 500 mg/kg bw/day. Under the study conditions, the maternal and developmental NOAEL of the test substance were 150 and 500 mg/kg bw/day, respectively (Edwards, 2010).