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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 14, 2009 to December 15, 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 408
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
The animal model, the Crl:CD(SD) rat, is recognized as appropriate for subchronic toxicity and reproduction studies. In addition, WIL Research Laboratories, LLC has extensive historical control data for subchronic and reproductive studies conducted using Crl:CD(SD) rat.
Sex:
male/female
Details on test animals and environmental conditions:
Sexually mature male and virgin female Crl:CD(SD) rats from Charles River Laboratories, Inc., Raleigh, NC, were used as the test system on this study. Rats were approximately 6.5 weeks old at experimental initiation and were sexually mature at the time of mating.

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
VEHICLE IDENTIFICATION
PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal) was used in preparation of the control and test diets.

PREPARATION
Diets containing the test substance were prepared on a weight/weight basis in the following manner for all animals. An appropriate amount of the test substance into tared glass jars and transferred into a Hobart mixer with 2500 grams of rodent feed (weight/weight). The formulation was mixed for 3 minutes; the resultant formulation was termed pre-mix. The remainder of the rodent feed used to achieve the desired concentration was weighed and placed in the Hobart mixer. The diet was mixed for 10 minutes to achieve a total batch of homogeneous diet at the appropriate concentration per test group.
Concentration of the test diet was based on mean body weight and food consumption for that group from the previous week in an attempt to administer a constant dose on a mg/kg/day level. The female test diets were calculated using mean body weight and food consumption data for the toxicology phase females or reproductive phase females as appropriate.
Concentration of test diets during mating, gestation, and lactation were determined based on the mean food consumption and body weight data for the reproductive phase females from the last week prior to mating (whether or not mating was confirmed). During the breeding period, males were fed the lower concentration test diet to prevent overexposure.

EXPOSURE
The control and test diets were offered ad libitum to the males and reproductive phase females for a minimum of 70 consecutive days prior to mating. The males continued to receive the control and test diets throughout mating until euthanasia. The reproductive phase females continued to receive the control and test diets throughout mating, gestation, and lactation until euthanasia. The toxicology phase females were offered the control and test diets ad libitum for at least 90 days until euthanasia. The males and reproductive phase females were exposed for the total of 130 and 113-128 consecutive days, respectively, and the toxicology phase females were exposed for a total of 91 consecutive days.
The offspring of the reproductive phase females were potentially exposed to the test diet in utero as well as via milk while nursing and via direct diet consumption of maternal test diet during the later portion of the pre-weaning period. Dietary exposure levels were selected based on the results of previous studies.
Details on mating procedure:
The males and reproductive phase females were paired on a 1:1 basis within each treatment group following 70 days of exposure to the test diet. All animals were randomly selected for pairing, avoiding sibling mating. A breeding record containing the male and female identification numbers and the start date of cohabitation was prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages. The males and reproductive phase females was mated once to produce one litter per generation (the F1 litters). Prior to pairing (Study Week 10), male body weights ranged from 404 g to 614 g and female body weights ranged from 190 g to 356 g. The animals were approximately 16 weeks old. For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity was determined on an 8 kg batch size at test diet concentrations of 50 and 13,000 ppm and room temperature stability for up to 10 days was determined on an 8 kg batch size at a test diet concentration of 13,000 ppm.
Samples for concentration analysis were collected weekly from the middle stratum of each formulation (including the control group). Concentration samples were analyzed for test substance concentration weekly for the first 4 weeks of the study and once a month thereafter for the remainder of the in-life phase.
Duration of treatment / exposure:
The test or basal diets were offered to all males for at least 70 days prior to mating and continued for at least 130 days until euthanasia. The appropriate diets were offered to the reproductive phase females for at least 70 days prior to mating and continued throughout mating, gestation, and lactation until euthanasia for a total of 113-128 days.
Details on study schedule:
28 April 2009.......................................... GLP experimental starting date (animal
receipt)
29 April 2009.......................................... Study initiation date (protocol signed by
Study Director)
14 May 2009........................................... Initiation of test diet exposure (experimental
start date)
14 May - 12 August 2009....................... Toxicology phase female test diet exposure
14 May - 18 September 2009 ................. Reproductive phase female test diet
exposure
14 May - 20 September 2009 ................. Male test diet exposure
22 July - 4 August 2009.......................... Mating period
23 July 2009............................................ First gestation day 0
13 August 2009....................................... Toxicology phase female necropsy
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.:
175 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five groups consisting of 12 males, 12 reproductive phase females, and 12 toxicology phase females
Control animals:
yes, concurrent no treatment
Details on study design:
The experimental design for this study consisted of a reproductive phase with four test-substance groups and one control group composed of 12 rats/sex each. In addition, a toxicology phase consisted of four test-substance groups and one control group composed of 12 females each. The selected animals were approximately 6 weeks old at the initiation of dietary substance exposure. Male body weight ranged from 160 g to 219 g, toxicology phase female body weights ranged from 145 g to 189 g, and reproductive phase female body weights ranged from 130 g to 172 g on the initial day of dietary substance exposure.

Examinations

Parental animals: Observations and examinations:
All reproductive phase females were allowed to deliver naturally and rear their young to PND 21. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia (difficulty in delivery).
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of estrus of each reproductive phase female for 21 days prior to pairing and continuing until evidence of mating was observed. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestus [D] from estrus [E] or proestrus [P], beginning 21 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation.
Litter observations:
On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

LITTER VIABILITY AND DEATHS

Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring dying were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Cannibalized pups were noted as such and discarded without further evaluation. Pups not expected to survive between birth and PND 4 to the next observation period (moribund) were euthanized via an intraperitoneal injection of sodium pentobarbital and necropsied. During PND 5 to weaning, moribund pups were euthanized by an intraperitoneal injection of sodium pentobarbital (prior to PND 11). A detailed gross necropsy was performed on any pup dying after PND 4; gross lesions were saved for possible future histopathological examination. The carcass of each pup was then discarded.

LITTER REDUCTION

To reduce variability among the litter, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. All selections were performed by computerized randomization. The remaining offspring were weighed, euthanized by intrathoracic injection of sodium pentobarbital and discarded on PND 4 without necropsy.

CLINICAL OBSERVATIONS

Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1, 4, 7, 10, 14, 17, and 21. Any abnormalities in nursing behavior were recorded.

BODY WEIGHTS

Pups were individually weighed on PND 1, 4, 7, 10, 14, 17, and 21. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data.

SEX DETERMINATION

Pups were individually sexed on PND 0, 4, 14, and 21.
Postmortem examinations (parental animals):
A complete necropsy was conducted on all animals euthanized in extremis or at the scheduled termination. The necropsy included examination of the external surface, all orifices and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The number of former implantation sites was counted for all reproductive phase females, and the number of unaccounted sites was calculated by subtracting the number of pups born from the number of implantation sites. Tissue collection in reproductive phase females: cervix, uterusa, mammary gland vagina ovaries with oviducts, all gross lesions (when possible), pituitary.
Postmortem examinations (offspring):
All surviving F1 rats were euthanized by carbon dioxide inhalation and necropsied on PND 21. Organs and tissues were saved in 10% neutral-buffered formalin for possible future histopathological examination only as deemed necessary by the gross findings. Representative specimens with external malformations not pertaining to the skeletal system were preserved in 10% neutral-buffered formalin.
Statistics:
All statistical analyses were conducted using SAS version 9.1 (SAS Institute, Inc., 2002-2003) software. Locomotor activity data were analyzed by BioSTAT Consultants, Inc., Portage, MI.
Reproductive indices:
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of estrus of each reproductive phase female for 21 days prior to pairing and continuing until evidence of mating was observed.
Offspring viability indices:
Each litter was examined daily for survival, and all deaths were recorded.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg/day group: reproductive phase females and included red material around the nose observed predominantly during the gestation and/or lactation periods. Yellow material around the urogenital area was occasionally noted during exposure for the 600 mg/kg/day males and reproductive phase females.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg/day: lower mean body weights and body weight gains (or losses) and reduced food consumption and food efficiency in both sexes generally throughout the exposure period. These effects were more prevalent for the males and reproductive phase females with body weights that were up to 16.4% and 34.3% lower than concurrent controls, respectively, versus 7.2% lower in the toxicology phase females and without a corresponding effect on food consumption.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The higher reticulocyte count noted in the 600 mg/kg/day toxicology phase females correlated with the higher mean cell volume and was consistent with increased erythropoiesis. Lower absolute and percent eosinophil counts were also noted in the 600 mg/kg/day group males; however, because the mean absolute value was within the historical control data, these differences were not considered adverse.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Developmental effects at the 600 mg/kg/day exposure level were evident as a lower mean number of implantation sites resulting in lower mean number of pups born and live litter size.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on reproductive performance, locomotor activity (no remarkable shifts in the pattern of habituation), or FOB evaluations (home cage, handling, open field, neuromuscular, and physiological observations) noted at any exposure level.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
175 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
other: reproductive
Organ:
ovary
pituitary gland
uterus
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:
not measured/tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean pup body weights in the 600 mg/kg/day group were slightly lower than the control group at birth and were approximately 56% lower than the concurrent control group on PND 21.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
175 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain

Results: F2 generation

Effect levels (F2)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects

Applicant's summary and conclusion

Conclusions:
Under the study conditions, there was no evidence of toxicity at exposure levels of 15, 50 and 175 mg/kg bw/day. Systemic toxicity was exhibited at 600 mg/kg bw/day by clinical findings, lower mean body weights, body weight gains and food consumption for both sexes, and lower absolute and relative-to-brain ovary, uterus and pituitary weights for the reproductive phase females. In addition, lower mean live litter size on PND 0, number of pups born and implantation sites and lower mean pup weights were noted in the 600 mg/kg bw/day group in the presence of excessive maternal toxicity. Therefore, the systemic, reproductive and developmental NOAEL was considered to be 175 mg/kg bw/day.
Executive summary:

A study was conducted to determine the potential adverse effects of the test substance when administered in the diet for at least 90 d and through one-generation of reproduction according to OECD Guidelines 408 and 422, in compliance with GLP. The study included evaluation of effects on male and female reproductive processes including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation, and on growth and development of the offspring through weaning. The males and reproductive phase females were bred to produce one litter. This study included five groups of Crl:CD(SD) rats consisting of 12 males, 12 reproductive phase females and 12 toxicology phase females. The treatment groups in each phase were offered a diet containing test substance at concentrations adjusted weekly to provide a target test substance consumption of 15, 50, 175 and 600 mg/kg bw/day. A concurrent control group received the basal diet on a comparable regimen. All animals were approximately 6 weeks of age at the initiation of diet exposure. The test or basal diets were offered to all males for at least 70 d prior to mating and continued for at least 130 d until euthanasia. The appropriate diets were offered to the reproductive phase females for at least 70 d prior to mating and continued throughout mating, gestation, and lactation until euthanasia for a total of 113-128 d. The toxicology phase females were euthanized after a maximum of 91 d of exposure to the appropriate diet. The summary of examinations and results of the clinical toxicity are included in repeated dose toxicity section. Vaginal lavages were performed daily for the reproductive phase females for determination of estrous cycles beginning 21 d prior to pairing. All reproductive phase females were allowed to deliver and rear their offspring to Lactation Day 21. F1 pups were necropsied on Postnatal Day (PND) 21. Each female in the reproductive and toxicology phases and all males received a complete detailed gross necropsy following the F1 pup necropsies or after at least 90 d of diet exposure; selected organs were weighed. Designated tissues from the males and toxicology phase females in the control and 600 mg/kg bw/day groups were examined microscopically. There was no test substance-related mortality or moribundity noted at any exposure level. However, test substance-related clinical findings were observed in the 600 mg/kg bw/day group reproductive phase females and included red material around the nose observed predominantly during the gestation and/or lactation periods. Yellow material around the urogenital area was occasionally noted during exposure for the 600 mg/kg/day males and reproductive phase females. Additionally, test substance-related lower absolute and relative-to-brain ovary, uterus, and pituitary weights were noted in the 600 mg/kg bw/day group reproductive phase females. There were no test substance-related effects on reproductive performance. Developmental effects at the 600 mg/kg bw/day exposure level were evident as a lower mean number of implantation sites resulting in lower mean number of pups born and live litter size. Additionally, mean pup body weights in the 600 mg/kg bw/day group were slightly lower than the control group at birth and were approximately 56% lower than the concurrent control group on PND 21. Postnatal survival was unaffected by test substance exposure and there were no test substance-related macroscopic findings noted for pups at necropsy. Under the study conditions, there was no evidence of toxicity at exposure levels of 15, 50 and 175 mg/kg bw/day. Systemic toxicity was exhibited at 600 mg/kg bw/day by clinical findings, lower mean body weights, body weight gains and food consumption for both sexes, and lower absolute and relative-to-brain ovary, uterus and pituitary weights for the reproductive phase females. In addition, lower mean live litter size on PND 0, number of pups born and implantation sites and lower mean pup weights were noted in the 600 mg/kg bw/day group in the presence of excessive maternal toxicity. Therefore, the systemic, reproductive and developmental NOAEL was considered to be 175 mg/kg bw/day (Edwards, 2010).