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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March 2009 to 10 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: Light, clear brown viscous liquid
- Storage condition of test material: The test substance was stored at room temperature, and was considered stable under this condition.
- Other: Documentation regarding the purity and stability of the test substance is on file with the Sponsor and WIL Research Laboratories, LLC. The Test Substance Certificate for the test substance was provided by the Sponsor.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sexually mature male and virgin female Crl:CD(SD) rats from Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: (P) approximately 7 wks
- Weight at study initiation: (P) Males: 219-266 g; Females: 150-183 g
- Housing: Following the initial acclimation period (F0) or selection (F1) and until pairing, all F0 and F1 parental test animals were housed individually, except during the mating period, in clean, stainless steel wire mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males remained housed in suspended wire mesh cages until the scheduled necropsy of the parental generation, and the females were transferred to plastic maternity cages with nesting material, ground corncob. The dams were housed in these cages until weaning on lactation day 21. Following weaning of the F1 litters, the F0 maternal animals were individually housed in suspended wire-mesh cages until the scheduled necropsy and the weaned F1 pups were housed together by litter for 1 week. Beginning on PND 28, the F1 pups were individually housed in suspended wire mesh cages until the start of the first mating period. Following weaning of the F2 litters, the F1 dams were returned to suspended wire-mesh cages until the start of the second mating period. Following weaning of the F2a litters, the F1 dams were individually housed in suspended wire-mesh cages until the scheduled necropsy. Females for which there was no evidence of mating were placed in plastic maternity cages with nesting material upon completion of the 14 day mating period. If these animals did not deliver after 25 days, they were returned to individual suspended wire-mesh cages.
- Diet (e.g. ad libitum): 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 and provided to WIL Research Laboratories, LLC. Feeders were changed and sanitized once per week. The basal diet was offered ad libitum to all groups throughout the acclimation period and to the control group (Group 1) throughout the study. Beginning study week 0, the appropriate test diet was offered to Groups 2-4 for at least 70 days prior to mating and throughout the mating and post-mating periods.
- Water (e.g. ad libitum): Reverse osmosis purified (on site) drinking water, delivered by an automatic watering system, was provided ad libitum throughout the acclimation period and during the study.
- Acclimation period: Animals were housed 3 per cage (by sex) in suspended wire-mesh cages for 3 days to allow for adaptation to the automatic watering system. Each animal 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 general changes in appearance and behavior.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6°C to 22.2°C
- Humidity (%): 42.0% to 56.6%
- Air changes (per hr): Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): Light timers were calibrated to provide a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod.
TEST ANIMALS
- Source: Sexually mature male and virgin female Crl:CD(SD) rats from Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: (P) approximately 7 weeks
- Weight at study initiation: (P) Males: 219-266 g; Females: 150-183 g
- Housing: Following the initial acclimation period (F0) or selection (F1) and until pairing, all F0 and F1 parental test animals were housed individually, except during the mating period, in clean, stainless steel wire mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males remained housed in suspended wire mesh cages until the scheduled necropsy of the parental generation, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. The dams were housed in these cages until weaning on lactation day 21. Following weaning of the F1 litters, the F0 maternal animals were individually housed in suspended wire-mesh cages until the scheduled necropsy and the weaned F1 pups were housed together by litter for 1 week. Beginning on postnatal day (PND) 28, the F1 pups were individually housed in suspended wire mesh cages until the start of the first mating period. Following weaning of the F2 litters, the F1 dams were returned to suspended wire-mesh cages until the start of the second mating period. Following weaning of the F2a litters, the F1 dams were individually housed in suspended wire-mesh cages until the scheduled necropsy. Females for which there was no evidence of mating were placed in plastic maternity cages with nesting material upon completion of the 14 day mating period. If these animals did not deliver after 25 days, they were returned to individual suspended wire-mesh cages.
- Diet (e.g. ad libitum): 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 and provided to WIL Research Laboratories, LLC. Feeders were changed and sanitized once per week. The basal diet was offered ad libitum to all groups throughout the acclimation period and to the control group (Group 1) throughout the study. Beginning study week 0, the appropriate test diet was offered to Groups 2-4 for at least 70 days prior to mating and throughout the mating and post-mating periods.
- Water (e.g. ad libitum): Reverse osmosis purified (on site) drinking water, delivered by an automatic watering system, was provided ad libitum throughout the acclimation period and during the study.
- Acclimation period: Animals were housed 3 per cage (by sex) in suspended wire-mesh cages for 3 days to allow for adaptation to the automatic watering system. Each animal 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 general changes in appearance and behavior.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 to 22.2°C
- Humidity (%): 42.0 to 56.6%
- Air changes (per hr): Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod (hrs dark / hrs light): Light timers were calibrated to provide a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod.

IN-LIFE DATES: From: To 10 March 2009 to 10 May 2010

Administration / exposure

Route of administration:
oral: feed
Vehicle:
acetone
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency):
The test diets were prepared approximately weekly

- Mixing appropriate amounts with (Type of food):
The test substance diets were prepared as mixtures in a fixed volume of acetone as follows. The appropriate amount of the test substance for each group was weighed into a tared, glass jar, and 200 mL of acetone was then added to the jar; the formulation was mixed using a magnetic stirrer until uniform. The test substance/acetone mixture was transferred into a Hobart mixer with 1 kg of rodent feed (weight/weight). A portion of the rodent feed was added to the jar; this was then scraped into the Hobart mixer to ensure complete transfer of the test substance/acetone mixture. The formulation was then mixed for 10 minutes. The resultant formulation was termed pre-mix. The remainder of rodent feed to achieve the desired concentration was weighed and placed in a V blender, and the pre-mix was then added to the blender. The diet was blended for 15 minutes (using an intensifier bar during the first and last 5 minutes) to achieve a total batch of homogeneous diet at the appropriate concentration per test group.

A separate batch of diets was prepared for males and females at each exposure level. During the breeding period, the test substance concentration in the diet was based on the lower concentration for each respective treatment group to prevent overexposure of the opposite sex. The initial diet concentrations were based on average food consumption and body weights during the pretest period. Diet concentrations were adjusted as necessary and were based on the mean body weight and food consumption values for each group (by sex) from the previous week to maintain the appropriate mg test substance/kg of body weight exposure, with the exception of the mating, gestation, and lactation periods, which were based on the pre-mating body weight and food consumption values. Amounts of the test substance used were not corrected for purity.
Details on the calculation for obtaining the correct doses in food are as follows:

1) Corrected mean bodyweight (g) = [((present mean bodyweight - previous mean bodyweight) x 4) / 7] + present mean bodyweight

2) Amount of test substance (mg) per kg of feed = [corrected mean bodyweight (kg) x exposure level (mg/kg/day)] / previous mean food consumption (g/animal/day)

3) Total amount of test substance required per sex per group = amount of test substance per kg of feed x 10

The concentration of the test substance in the diet for the F1 pups during the first 2 weeks following weaning were calculated based on historical control pup body weight and food consumption data.


- Storage temperature of food:
The test diets were placed in labeled bags and stored at room temperature and were considered stable under these conditions.

VEHICLE
PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 mixed with acetone was used in the preparation of the control (basal) and test diets.

- Lot/batch no. (if required):
Acetone (lot nos. XQ0253, WQ0308, YR0479, and YF0308, exp. dates: 6 November 2009, 13 May 2010, 27 April 2011, and 27 April 2011, respectively, received from Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ or Gardena, CA)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: The rats were paired for mating in the home cage of the male for up to 14 days.
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in 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.
- Further matings: When evidence of mating was not apparent after 14 days, the female was placed in a plastic maternity cage with nesting material, with no further opportunity for mating.
- After successful mating each pregnant female was caged (how): The animals were separated, and the female was housed in an individual plastic cage with nesting material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of test substance exposure, a batch of the control diet and batches of the test diet admixes at both the lowest and highest projected admixture concentrations to be used on the study (7 and 2000 ppm, respectively) were prepared. Duplicate samples (100 g) were collected from the middle of the control diet and top, middle, and bottom of the 7 and 2000 ppm diets. One set of samples at each dietary concentration was analyzed for homogeneity. The other set of samples collected from each diet was analyzed for 8- and 15-day stability under laboratory conditions. Samples (100 g) from the middle of the control and test diet formulations were collected weekly throughout the study. During the first 4 weeks of the study, and once per month for the remainder of the in-life phase, these weekly samples were analyzed for concentration. All other weekly samples that were collected, but not analyzed for concentration, were purged with nitrogen and stored frozen. All analyses were performed by the Analytical Chemistry Department, WIL Research Laboratories, LLC.

RESULTS OF ANALYSIS
The dietary formulations were within the WIL Research Laboratories, LLC’s standard operating procedures range for dietary admixes (85 to 115%), were homogeneous and were stable for up to 15 days at room temperature.
Duration of treatment / exposure:
F0 males and females were exposed for 129-134 consecutive days and F1 males and females were directly exposed for 210-227 consecutive days.
Frequency of treatment:
The control and test diets were offered ad libitum to the F0 and F1 males and females for a minimum of 70 consecutive days prior to mating. The F0 and F1 males continued to receive the test and control diets throughout mating and through the day of euthanasia. The F0 and F1 females continued to receive the control and test diets throughout mating, gestation, and lactation through the day of euthanasia.
Details on study schedule:
The F1 animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of exposure. All animals were randomly selected for pairing, avoiding sibling matings. After a minimum of 26 days following euthanasia of the F2 pups on PND 21, each F1 male was paired with the same F1 female for a second mating period.

The F1 generation was mated twice to produce a maximum of 2 litters per dam (the F2 and F2a litters). Immediately prior to the initial F1 pairing (study week 28), male body weights ranged from 337 to 714 g and female body weights ranged from 233 to 387 g; the animals were 13-15 weeks old. Immediately prior to the second F1 pairing (study week 39), male body weights ranged from 411 to 963 g and female body weights ranged from 257 to 476 g; the animals were 24-26 weeks old.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1.5, 15 & 75 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
30 rats/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
Exposure levels were determined from the results of previous studies and were provided by the Sponsor Representative after consultation with the Study Director. The high exposure level of 75 mg/kg/day was selected based on the effects observed in a 90-day dietary dose range-finding study of the test substance performed by the same laboratory and reported elsewhere. In that study, decreased body weight gain was observed at 50, 100, 150, and 200 mg/kg/day and decreased absolute weights of reproductive organs were observed at 100, 150, and 200 mg/kg/day.
The mid-exposure level of 15 mg/kg/day was selected based on a 5-fold decrease from the high exposure level. At this exposure level, minimal toxicity was expected based on findings observed at 25 mg/kg/day in a previous one generation oral (gavage) study of the test substance also performed by the same laboratory and reported elsewhere.
The low exposure level of 1.5 mg/kg/day was selected based on a 10-fold decrease from the mid-exposure level. At this exposure level, a no-observed-effect level was expected to be obtained based on a comparison of the results observed at 5 mg/kg/day in the one-generation study.
Positive control:
No data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were recorded weekly for all parental animals throughout the study period.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual F0 and F1 male body weights were recorded weekly throughout the study and prior to the scheduled necropsy. Individual F0 and F1 female body weights were recorded weekly until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating treatment period (F0 and F1 females and F0 males) and for the entire treatment period (F0 and F1 males). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1, 4, 7, 14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Individual F0 and F1 male and female food consumption was measured weekly until pairing. Food intake was not recorded during the mating period. Following mating, F0 and F1 male food consumption was measured on a weekly basis until the scheduled necropsy or second pairing, respectively. Following the second mating, F1 male food consumption was measured on a weekly basis until the scheduled necropsy. Female food consumption was recorded daily during gestation and lactation.
- 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
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of estrous of each F0 and F1 female for 21 days prior to pairing and continuing until evidence of mating was observed or until the end of the mating period; for F1 females, this procedure was done only during the first mating cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [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. Vaginal lavages were also performed on the day of necropsy to determine the stage of estrous
Sperm parameters (parental animals):
Parameters examined in F0/F1 male parental generations:
Immediately upon euthanasia, the reproductive tract of each F0 and F1 male was exposed via a ventral mid line incision. The right testis and epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL bovine serum albumin (BSA). After a 10 minute incubation period, a sample of sperm was loaded into a 100 µm cannula for determination of sperm motility.
Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique (Linder et al., 1992). Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
Litter observations:
STANDARDISATION OF LITTERS
To reduce variability among the litters, 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 euthanized by intraperitoneal injection of sodium pentobarbital and discarded on PND 4.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 / F2a offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioral abnormalities, developmental landmarks, balanopreputial separation & vaginal patency.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

PARTURITION
Beginning 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 was first observed.
Postmortem examinations (parental animals):
SACRIFICE
All surviving F0 adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation. All surviving F1 adults were euthanized by carbon dioxide inhalation following weaning of the F2a pups.

GROSS NECROPSY
A complete necropsy was conducted on all parental animals (F0 and F1) found dead, euthanized in extremis, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera.
For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each F0 female by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females necropsied during gestation through lactation day 4. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy. The following F0 and F1 parental tissues and organs were collected and were placed in 10% neutral buffered formalin:

Adrenal glands, Aorta, Bone with marrow (sternebrae), Brain (Cerebrum level 1 and 2, Cerebellum with medulla/pons), Cervix, Coagulating glands, Eyes with optic nerve, Gastrointestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Heart, Kidneys, Liver (sections of 2 lobes), Lungs (including bronchi), Lymph node (mesenteric), Ovaries and oviducts, Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Salivary gland (mandibular), Seminal vesicles, Skeletal muscle (rectus femoris), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymides and vas deferens, Thymus gland, Thyroids (with parathyroids if present), Trachea, Urinary bladder, Uterus with vagina, all gross lesions.

ORGAN WEIGHTS
Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight and/or brain weight ratios were reported.
The following organs were weighed from all F0 and F1 parental animals at the scheduled necropsies:
Adrenal glands Prostate gland
Brain Seminal vesicles with coagulating glands (with accessory fluids)
Epididymides (total and cauda) Spleen
Kidneys Testes (These paired organs were weighed separately)
Liver Thymus gland
Ovaries Thyroid gland
Pituitary gland Uterus with oviducts and cervix

HISTOPATHOLOGY
Microscopic evaluations were performed on the following tissues for all F0 and F1 parental animals from the control and high exposure groups and for all adult animals found dead and euthanized in extremis.
Adrenal glands Prostate gland
Brain Seminal vesicles
Cervix Spleen
Coagulating gland Testis (right)
Epididymis, right (caput, corpus & cauda) Thymus gland
Kidneys Uterus
Liver Vagina
Ovaries Vas deferens
Oviducts All gross (internal) lesions (all groups)
Pituitary gland

In addition, microscopic evaluations were performed on the ovaries from the F0 and F1 females and the kidneys from the F0 and F1 males in the low- and mid-exposure groups, and the reproductive organs of all animals suspected for reduced fertility (e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected).
Postmortem examinations (offspring):
SACRIFICE
All remaining nonselected F1, F2, and F2a weanlings were euthanized by carbon dioxide inhalation and necropsied on PND 21.

GROSS NECROPSY
Gross necropsies with emphasis on developmental morphology and organs of the reproductive system were performed on nonselected F1, F2, and F2a pups euthanized by carbon dioxide inhalation on PND 21. Selected F1, F2, and F2a organs (brain, spleen, and thymus) were collected from 1 pup/sex/litter that survived to the scheduled termination on PND 21. These tissues and all gross lesions from F1, F2, and F2a weanlings were preserved in 10% neutral buffered formalin for possible future histopathologic examination; all other tissues and the carcasses were discarded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Tissues and all gross lesions from F1, F2, and F2a weanlings were preserved in 10% neutral buffered formalin for possible future histopathologic examination. The brain, spleen, and thymus were weighed from 1 pup/sex/litter at the scheduled necropsies on PND 21.
Statistics:
Analyses were conducted using two tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance exposed group to the control group by sex.

Parental mating, fertility, copulation, and conception indices were analyzed using the Chi square test with Yates’ correction factor.
Mean parental and offspring body weights and body weight changes, parental food consumption, and food efficiency data, estrous cycle lengths, pre coital intervals, gestation lengths, former implantation sites, live litter sizes, unaccounted for sites, numbers of pups born, anogenital distance, balanopreputial separation data, vaginal patency data, absolute and relative organ weights, sperm production rates, epididymal and testicular sperm numbers, and ovarian primordial follicle counts were subjected to a parametric one way analysis of variance (ANOVA) to determine intergroup differences between the control and test substance exposed groups.
If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance exposed groups to the control group.
Mean litter proportions of postnatal pup survival and pup sexes at birth, percentages of motile and progressively motile sperm and percentages of sperm with normal morphology were subjected to the Kruskal Wallis nonparametric ANOVA test to determine intergroup differences between the control and test substance exposed groups.
If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance exposed groups to the control group.
Histopathological findings in the test substance exposed groups were compared to the control group using a two tailed Fisher’s Exact test.
Reproductive indices:
- Male (Female) Mating Index (%)
- Male Fertility Index (%)
- Male Copulation Index (%)
- Female Fertility Index (%)
- Female Conception Index (%)
Offspring viability indices:
- Mean Live Litter Size
- Postnatal Survival Between Birth and PND 0 or PND 4 (Pre Selection) (% Per Litter)
- Postnatal Survival for All Other Intervals (% Per Litter)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test substance-related mortalities in any F0 parental animals at any exposure level. However 2, 1 and 1 F0 animals in the 1.5, 15 and 75 mg/kg/day dose groups respectively were found dead or euthanized in extremis during the generation.
Test substance-related clinical findings of decreased defecation and small feces were noted for F0 females in the 75 mg/kg/day group beginning study week 7 and continuing through gestation day 13; these findings corresponded to reduced food consumption in this group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the 75 mg/kg/day group, test substance-related, lower body weights, body weight gains, food consumption, and food efficiency were generally noted throughout the entire generation for the F0 and F1 males, and throughout the pre-mating period for the F0 females. For the F1 females in the 75 mg/kg/day group, lower body weights, body weight gains (or absence of body weight gain or body weight losses), and food consumption were generally noted throughout the first and second pre-mating periods and following the second mating; however, mean food efficiency in the 75 mg/kg/day group F1 females were unaffected by test substance exposure during these time periods.
Lower mean body weights continued for the 75 mg/kg/day group F0 and F1 females during each gestation and lactation period due to the decrements noted during the pre mating periods. Mean body weight gains in 75 mg/kg/day group F0 and F1 females were unaffected by test substance exposure for the overall gestation period. During lactation, a mean body weight loss was noted in the 75 mg/kg/day group F0 females during lactation days 1-4; however, this reduction was not noted in the F1 females during either lactation period. During the latter portion of each lactation period, mean body weight gains in the 75 mg/kg/day group F0 and F1 females were higher than the control group; these higher body weight gains were attributed to the developmental delay of the F1, F2, and F2a pups.

Mean body weights, body weight gains, food consumption, and food efficiency for F0 and F1 males and females at 1.5 and 15 mg/kg/day were unaffected by test substance exposure.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Mean food consumption (generally g/animal/day) in the 75 mg/kg/day group F0 and F1 females was lower than the control group for the overall gestation and lactation periods. Mean food efficiency in the F0 and F1 females at 75 mg/kg/day during each gestation and lactation period was generally higher than the control group.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Exposure at 75 mg/kg/day resulted in longer mean estrous cycle lengths in F0 and F1 females. Mean estrous cycle lengths for F0 and F1 females at 1.5 and 15 mg/kg/day, and reproductive indices for F0 and F1 males and females at 1.5, 15, and 75 mg/kg/day were unaffected by test substance exposure.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The mean epididymis sperm concentration for F0 males in the 75 mg/kg/day group was lower than the control group. There were no effects on spermatogenic endpoints for F0 males at 1.5 and 15 mg/kg/day or F1 males at 1.5, 15, and 75 mg/kg/day.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no test substance-related effects on F0 or F1 gestation lengths, the process of parturition, or the mean number of days between pairing and coitus at any exposure level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Test substance-related organ weight changes at 75 mg/kg/day consisted of lower weights of the left and right epididymides and cauda epididymides, prostate, and seminal vesicles/coagulating glands, and a higher pituitary weight in F0 and F1 males, lower left and right testes weights in F1 males, lower ovary weights in F0 and F1 females, and a higher adrenal gland weight in F1 females. There were no test substance-related organ weight changes for F0 and F1 males and females at 1.5 and 15 mg/kg/day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related gross necropsy observations for F0 and F1 males and females at any exposure level.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test substance-related histopathologic changes were limited to renal mineralization in the 75 mg/kg/day group F0 males and 15 and 75 mg/kg/day group F1 males and decreased corpora lutea in the 75 mg/kg/day group F0 and F1 females.

OTHER FINDINGS (PARENTAL ANIMALS)
A decrease in the mean number of former implantation sites was noted in the 75 mg/kg/day group F0 females compared to the control group; this resulted in a slightly lower mean number of pups born and live litter size in this group. Because the F1 females were mated twice, the mean number of implantation sites for these females could not be calculated for each mating. However, slightly lower mean numbers of pups born and live litter sizes were noted for the F2 and F2a litters at 75 mg/kg/day.

Effect levels (P0)

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Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
15 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects body weight; food consumption organ weights; histopathology.
Remarks on result:
other: Generation: F0 (migrated information)
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on decreased implantation sites, increased estrous cycle lengths and a reduction in mean epididymal sperm concentration.
Remarks on result:
other: Generation: F0 and F1 (migrated information)
Dose descriptor:
NOAEL
Remarks:
Neonatal toxicity
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
The percentages of males at birth and the general physical condition of the pups in the 1.5, 15, and 75 mg/kg/day groups were unaffected by exposure to the test diet in the F0 and F1 generations. There were no effects on postnatal survival during the pre-weaning period for F1 pups at any exposure level. However, in the F2 and F2a pups, reductions in postnatal survival were noted from birth to PND 4 (pre-selection) at 75 mg/kg/day. Postnatal survival of the F2 and F2a pups during the pre-weaning period at 1.5 and 15 mg/kg/day was unaffected by parental test substance exposure.

CLINICAL SIGNS (OFFSPRING)
Two F1 males and 3 F1 females that were selected for the F1 generation were found dead or euthanized in extremis shortly following weaning (PND 23-30); however, these deaths were attributed to the test substance related effects on pre-weaning body weights, as the deaths occurred in all low-weight pups (13.7 g to 25.2 g on PND 21). There were no test substance-related mortalities in any of the F1 parental animals at any exposure level during the remainder of the generation. No remarkable clinical findings were noted at any exposure level in the F1 generation.

BODY WEIGHT (OFFSPRING)
In the F1 male and female pups, lower mean body weight gains were noted throughout the pre-weaning period (PND 1-21) at 75 mg/kg/day, resulting in lower (7.7% to 30.2%) mean body weights from PND 4-21 compared to the control group. In the F2 male and female pups, lower (9.5% and 10.0%, respectively) mean birth weights were noted at 75 mg/kg/day, and lower mean body weights (6.7% to 21.2% and 8.1% to 20.6%, respectively) and body weight gains were noted at this exposure level from PND 4-21. In the F2a male and female pups, lower mean body weight gains were noted in the 75 mg/kg/day group beginning on PND 4 and 7, respectively, through PND 21. Mean body weights in the 75 mg/kg/day group F2a pups were lower than the control group from PND 7-21 for males (8.9% to 19.4%) and 14-21 for females (12.6% to 15.8%). Mean F1, F2, and F2a pup body weights at 1.5 and 15 mg/kg/day were unaffected by test substance exposure.

SEXUAL MATURATION (OFFSPRING)
The aforementioned lower mean body weights in the 75 mg/kg/day group F1 males (prior to and following weaning) resulted in a delay in the attainment of balanopreputial separation.
Conversely, F1 females in the 75 mg/kg/day group attained vaginal patency at an earlier age than females in the control group.
The attainment of sexual developmental landmarks for F1 males and females at 1.5 and 15 mg/kg/day were unaffected by test substance exposure. There were no test substance-related effects on F2 anogenital distance at any exposure level.

ORGAN WEIGHTS (OFFSPRING)
Mean absolute and/or relative (to final body weight) spleen and thymus gland weights in the F1, F2, and F2a pups at 75 mg/kg/day were lower than the control group on PND 21; however, these organ weight changes were considered secondary to the test substance-related effect on pup body weight gain at this exposure level that resulted in lower mean pup body weights. There were no test substance-related effects on F1, F2, and F2a pup organ weights at 1.5 and 15 mg/kg/day.

GROSS PATHOLOGY (OFFSPRING)
There were no F1, F2, or F2a pup necropsy findings that could be attributed to parental exposure to the test substance at any exposure level.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Generation:
F1
Effect level:
1.5 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects body weight; food consumption organ weights; histopathology.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Parental toxicity was evidenced by lower mean body weights, body weight gains, and food consumption in the F0 and F1 males and females in the 75 mg/kg/day group. In addition, several organ weight changes (lower weights of the cauda epididymides, epididymides, prostate, and seminal vesicles/coagulating glands, and higher pituitary weight for F0 and F1 males; lower left and right testes weight for F1 males; lower ovary weights for F0 and F1 females; and higher weight of adrenal glands for F1 females) were noted for parental animals at 75 mg/kg/day. Furthermore, histopathologic changes of renal mineralization in F0 males at 75 mg/kg/day and F1 males at 15 and 75 mg/kg/day, as well as decreased corpora lutea in F0 and F1 females at 75 mg/kg/day were noted. Therefore, the no-observed-adverse-effect level (NOAEL) for F0 and F1 parental toxicity was considered to be 15 and 1.5 mg/kg/day, respectively.

Decreased implantation sites (F0 females), increased estrous cycle lengths (F0 and F1 females), and a reduction in mean epididymal sperm concentration (F0 males) were noted at 75 mg/kg/day. Therefore, the NOAEL for male and female reproductive toxicity was considered to be 15 mg/kg/day.

Based on reductions in F2 and F2a postnatal survival, lower F1, F2, and F2a offspring body weights and body weight gains (that resulted in a delay in the mean age of balanopreputial separation, lower spleen and thymus weights, and post-weaning mortality) and the accelerated onset of vaginal patency in F1 females at 75 mg/kg/day, the NOAEL for neonatal toxicity was considered to 15 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
The no-observed-adverse-effect level (NOAEL) for F0 and F1 parental toxicity was considered to be 15 and 1.5 mg/kg/day, respectively.
The NOAEL for male and female reproductive toxicity was considered to be 15 mg/kg/day.
The NOAEL for neonatal toxicity was considered to 15 mg/kg/day.
Executive summary:

The reproductive toxicity of tetrapropenyl phenol was examined in a two-generation reproductive toxicity study in rats. The study was conducted in accordance with OECD Guideline 416 and to GLP standards.

Three groups of male and female Crl:CD(SD) rats (30/sex/group) were offered the test substance continuously in the diet for at least 70 consecutive days prior to mating. Target test substance concentrations were 1.5, 15, and 75 mg/kg/day for the F0 and F1 generations. A concurrent control group (30 rats/sex) was offered the basal diet continuously throughout the study. F0 males and females were exposed for 129 - 134 consecutive days, and F1 males and females were exposed for 210 - 227 consecutive days.

All animals were observed twice daily for appearance and behavior. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for males throughout the study and for females prior to mating and during gestation and lactation.

Parental toxicity was evidenced by lower mean body weights, body weight gains, and food consumption in the F0 and F1 males and females in the 75 mg/kg/day group. In addition, several organ weight changes (lower weights of the cauda epididymides, epididymides, prostate, and seminal vesicles/coagulating glands, and higher pituitary weight for F0 and F1 males; lower left and right testes weight for F1 males; lower ovary weights for F0 and F1 females; and higher adrenal glands weight for F1 females) were noted for parental animals at 75 mg/kg/day. Furthermore, histopathologic changes of renal mineralization in F0 males at 75 mg/kg/day and F1 males at 15 and 75 mg/kg/day, as well as decreased corpora lutea in F0 and F1 females at 75 mg/kg/day were noted. Therefore, the no-observed-adverse-effect level (NOAEL) for F0 and F1 parental toxicity was considered to be 15 and 1.5 mg/kg/day, respectively.

Decreased implantation sites (F0 females), increased estrous cycle lengths (F0 and F1 females), and a reduction in mean epididymal sperm concentration (F0 males) were noted at 75 mg/kg/day. Therefore, the NOAEL for male and female reproductive toxicity was considered to be 15 mg/kg/day.

Based on reductions in F2 and F2a postnatal survival, lower F1, F2, and F2a offspring body weights and body weight gains (that resulted in a delay in the mean age of balanopreputial separation, lower spleen and thymus weights, and post-weaning mortality) and the accelerated onset of vaginal patency in F1 females at 75 mg/kg/day, the NOAEL for neonatal toxicity was considered to 15 mg/kg/day.