Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility was evaluated in an OECD 443 extended 1 -generation study. There was no effect of the test article on fertility in male or female rats.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes
Limit test:
no
Justification for study design:
The study design was based on the OECD 443 guideline, and the specifics after consultation with the EU authorities who ordered the testing. Further decisions on additional work were determined by the information available to the participants at the time of the result evaluation.
Specific details on test material used for the study:
Test Material Name Biphenyl
Chemical Name 1,1’-Biphenyl
Lot/Reference/Batch Number 5M009
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.79 ± 0.00% (corrected for water) by gas chromatography with identification by gas
chromatography/mass spectrometry and Fourier transform infrared spectroscopy (Ferruzzi, 2016). Test Material Stability Under Storage
Conditions Biphenyl, lot 5M009, was determined to be stable for 24 months under ambient storage conditions as tested under U.S. EPA
OPPTS Guideline 830.6317 (Ferruzzi and Palumbo, 2016).
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Crl:CD(SD) (Sprague-Dawley) rat has been shown to be an appropriate animal model for this study design
Sex:
male/female
Details on test animals and environmental conditions:
Species and Sex: Rats, (male and female)
Strain and Justification: Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location Charles River Laboratories (Raleigh, North Carolina)
Age at Study Start 8 weeks at initiation of treatment
Health Status and Acclimation Upon arrival all animals were acclimated to the laboratory for approximately one week prior to the study. During the acclimation period, each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
Housing
Upon arrival, animals were housed two to three per cage in stainless steel cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated lixit valve-type watering system. After assignment to the study, animals were single housed in solid bottom stainless steel cages, except during the breeding, gestation, and littering phases of the study and from weaning until PND 28. The solid bottom cages contained ground corn cob bedding. During breeding, animals were placed in stainless steel cages with wire mesh floors that were suspended above absorbent paper in order to better visualize copulatory plugs. During gestation and littering, dams (and their litters) were housed in plastic cages provided with irradiated ground corn cob bedding and paper nesting material from approximately GD 0 until completion of lactation (LD 21). Selected F1 offspring were housed in plastic cages with same sex littermates from PND 21-28. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were targeted in the animal room from the day of arrival till necropsy; however, temporary excursions from these environmental conditions may have occurred on an infrequent basis; all observed ranges were documented in the study file.

Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.) Photoperiod times may have changed due to study-related activities.


Enrichment Enrichment for animals was given from the day of arrival until necropsy. Enrichment included the use of open areas on the cage sides for visualization of other rats (except when in wire-bottom cages). In addition, the cage contained nylon bones or paper nesting material (during the gestation and lactation phases of the study).

Feed and Water Feed and municipal water were provided ad libitum. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained in the study file. Results of the feed and water analyses indicated no contaminants that would interfere with the conduct of the study or the interpretation of the results.
Route of administration:
oral: feed
Details on exposure:
Diet:

Diets (LabDiet Certified Rodent Diet #5002) were mixed by serially diluting a concentrated test material-feed mixture (premix) with ground feed. A Quadro Co-mil was used during preparation of the premix to facilitate homogeneous distribution of the test material. Diets were prepared as a fixed percent in rodent feed. The concentration of the diets were not adjusted for purity and were prepared periodically based on stability data

Analysis

Concentration Verification and Homogeneity

Dose confirmation analysis of all dose levels, plus control and premix were determined during the pre-breeding and during the lactation phase and during adulthood in the F1 offspring. The homogeneity of the low-dose and the high-dose diets was determined concurrent with dose confirmation. Analysis was performed using gas chromatography (GC) with mass spectrometry (MS) detection. The method used was based on the previously validated method with appropriate modifications as needed to meet the study requirements. Details of the analyses were maintained in the study files.

Stability

Biphenyl was shown to be stable in rodent feed for 21 days at concentrations ranging from 0.0005-10%. At a concentration of 10%, 35 days of stability was established. The concentration ranges tested in the stability study spanned the concentrations planned for this study, and mixed diets were used within the established stability limits.
Details on mating procedure:
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Breeding Procedure

Breeding of the P1 adults commenced after approximately 10 weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating). Animals were paired until mating occurred or two weeks had elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and placed in a plastic cage with irradiated corn cob bedding. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. Breeding of the Cohort 1B F1 offspring was triggered; therefore, one male was paired with one female for 2 weeks beginning on approximately PND 120. Cohabitation of male and female littermates was avoided.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration Verification and Homogeneity

Dose confirmation analysis of all dose levels, plus control and premix were determined during the pre-breeding and during the lactation phase and during adulthood in the F1 offspring. The homogeneity of the low-dose and the high-dose diets was determined concurrent with dose confirmation. Analysis was performed using gas chromatography (GC) with mass spectrometry (MS) detection.

Biphenyl was shown to be stable in rodent feed for 21 days at concentrations ranging from 0.0005-10%. At a concentration of 10%, 35 days of stability was established. The concentration ranges tested in the stability study spanned the concentrations planned for this study, and mixed diets were used within the established stability limits.
Duration of treatment / exposure:
Food with test article was provided ad libitum
Frequency of treatment:
daily
Details on study schedule:
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Experimental Design and Critical Dates

Groups of 26 male and 26 female Crl:CD(SD) rats/dose were fed diets supplying approximately 0, 300, 1000, or 2800 ppm biphenyl for approximately ten weeks prior to breeding and continuing through breeding (up to two weeks), approximately 7 additional weeks (males) or gestation (three weeks) and lactation (three weeks) for females. P1 females were exposed until LD 22 (the end of the lactation period). After weaning, one male and one female F1 pup/litter, if possible, were randomly assigned to cohorts 1A & 1B and one male or one female F1 pup/litter were randomly assigned to cohorts 2A, 2B, and 3 as follows:

• Cohort 1A: Assessment of effects on reproductive systems and toxicity (≥20M+20F/dose).
• Cohort 1B: Assessment of effects on reproductive systems and toxicity (≥20M+20F/dose).
• Cohort 2A: Assessment of DNT post weaning (11-12M+11F/dose).
• Cohort 2B: Assessment of DNT at weaning (10M+10F/dose).
• Cohort 3: Assessment of DIT (10M+10F/dose).

In addition, unselected control weanlings were assigned as positive controls to cohorts 1A and 3 as follows:
• Cohort 1A: Splenic lymphocyte positive control (10M).
• Cohort 3: Immunotoxicity positive control (IPC) (5M+5F).

The positive control animals were excluded from study specific parameters unless specified.

Selected F1 offspring were maintained on the test diet until PND 22 (Cohort 2B), PND 78 (Cohort 2A), ~PND 90 (Cohort 1A), ~PND 120 (Cohort 1B), or ~PND 56 (Cohort 3). Reproductive and selected data from the P1 generation and selected reproductive and toxicity data from the F1 generation were used to trigger a second generation. A comprehensive evaluation of male and female reproductive systems were conducted, and included an evaluation of gonadal function, the estrous cycle, mating performance, conception, gestation, parturition and lactation, as well as survival, growth, and development of the offspring. Selected systemic toxicity parameters were also evaluated in the P1 and P2 males and females. Offspring were evaluated for effects on the nervous system, reproductive system, immunotoxicity and other systemic toxicity parameters.

A satellite group of P1 females (4/dose) were included primarily for assessments of kidney function in adult non-pregnant females during the pre-breeding period based on the probe and rangefinder study findings. The satellite females were excluded from study specific parameters unless specified. Test material administration began on February 24, 2017. The satellite females were necropsied on May 08, 2017. The P1 females were necropsied from June 19, 2017 to July 02, 2017. The P1 males were necropsied from July 10, 2017 to July 13, 2017. Cohort 1A animals were necropsied from August 25, 2017 to September 07, 2017. Cohort 1B P2 females were necropsied from November 06, 2017 to November 18, 2017. The Cohort 1B P2 males were necropsied from October 23, 2017 to October 26, 2017. Cohort 2A animals were necropsied from August 14, 2017 to August 27, 2017. Cohort 2B animals were necropsied from June 19, 2017 to June 28, 2017. Cohort 3 animals were necropsied from July 24, 2017 to August 02, 2017. The F1 unselected weanlings were necropsied from June 19, 2017 to July 02, 2017. The F2 unselected weanlings were necropsied from November 06, 2017 to November 18, 2017.
Dose / conc.:
300 ppm
Remarks:
~ 25 mg/kg nominal
Dose / conc.:
1 000 ppm
Remarks:
~ 75 mg/kg nominal
Dose / conc.:
2 800 ppm
Remarks:
~ 215 mg/kg nominal
No. of animals per sex per dose:
26
Control animals:
yes, plain diet
Details on study design:
Route, Method of Administration, Frequency, Duration and Justification

The dietary route was required by the CoRAP decision of the Portuguese and ECHA Member State Committee competent authorities. Compared to gavage administration, additional advantages of the dietary route of exposure include the provision of a stable and consistent (steady state) systemic exposure throughout all stages of embryo/fetal development. reductions in stress, avoidance of potential vehicle-induced confounding effects, and the elimination of potential dosing-related injuries to the maternal animals. Thus, oral administration of the test material in feed represents an appropriate means of the test material exposure.

Dose Levels and Justification

Dose level selection for the biphenyl extended one-generation reproductive toxicity test considered: body weight, body weight gain, and feed consumption decreases, organ weight increases with associated histopathology, pup weights and toxicokinetics from a previous study to establish the high dose of 2800 ppm. The selected dose levels were selected using a traditional maximum tolerated dose (MTD) approach based primarily on the results of the OECD 421 reproductive toxicity screening study. Based on this study, dietary exposure to the high dose of 2800 ppm (215 mg/kg/day) was expected to result in effects on body weight, feed consumption, liver and kidney weights in males and/or females and effects on body weights in the offspring during lactation. Linear kinetics were observed for the biphenyl in females, pups and milk on LD 10 at doses at or above 1375 ppm in the OECD 421 study. The mid- and low- dose levels were selected to provide possible dose-response data for any observed treatment-related effects in the high-dose group. The low-dose was expected to be a NOAEL.
Parental animals: Observations and examinations:
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Daily Observations
A cage-side examination was conducted on all animals (including satellite females and positive control animals) at least twice daily with one exception. A PM daily cage-side observation was inadvertently not conducted on June 02, 2017. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could have been observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Cage-side examinations were also conducted on dams and their litters at least twice daily.

Detailed Clinical Observations
Detailed clinical observations (DCO) were conducted on all P1 study animals pre-exposure and weekly throughout the pre-breeding (including satellite females) and breeding periods. DCO were conducted on all P2 study animals weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received detailed clinical examinations on GD 0, 7, 14, and 21. Females that delivered litters were subsequently evaluated on LD 1, 7, 14, and 21. Detailed clinical observations may not have been conducted on females that failed to mate or deliver a litter during the gestation and lactation phases of the study. F1 offspring also were given weekly DCO evaluations after weaning with one exception. A PND 77 detailed clinical observation was inadvertently not conducted for Cohort F1 2A animals 1804 and 1828. The DCO was conducted at approximately the same time each examination day, according to an established format. The examination included cage-side, hand-held, and open-field observations, which were recorded categorically or using explicitly defined scales (ranks).
Oestrous cyclicity (parental animals):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Vaginal lavage samples from all P1 and P2 females were collected daily for 2 weeks prior to mating and during cohabitation until each female was sperm- or plug-positive or until the two week mating period has elapsed. Lavage samples were collected by gently irrigating the vagina with water and transferring lavage fluid to a microscope slide. Vaginal lavage slides were examined microscopically to determine estrous cycle length and pattern. Vaginal smears were collected in Cohort 1A F1 offspring after vaginal opening until the first cornified smear was recorded to determine the time interval between vaginal patency and first estrus. The estrous cycle also was evaluated in Cohort 1A F1 females for 2 weeks from approximately PND 75 through PND 88. On the day of scheduled necropsy, the stage of the estrous cycle was determined for all P1 and P2 and adult F1 female rats in Cohort 1A.
Sperm parameters (parental animals):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Sperm parameters were evaluated in P1, P2, and Cohort 1A F1 males at termination. Unless circumstances dictate otherwise, the left and right epididymides and testes were allocated as follows: right epididymis – motility and histopathology; left epididymis – counts; right testis – histopathology; left; testis – counts.

Motility
Sperm motility was evaluated in P1, P2, and Cohort F1 males from all dose groups. Immediately after euthanasia of males and isolation of their epididymides, a small sample of sperm from the right cauda epididymis was expressed into a dish containing pre-warmed Modified Sperm Washing Medium (Irvine Scientific, Santa Ana, California) and was incubated for approximately 2-3 minutes. An aliquot of the incubated sperm suspension was placed on a slide and loaded into the Hamilton-Thorne Integrated Visual Optical System (IVOS; Hamilton-Thorne Research, Beverly, Massachusetts) for the determination of total percent motile (showing any motion) and percent progressively motile (showing net forward motion) sperm. After sperm were released, the epididymis was placed in Bouin’s fixative and subjected to histopathologic examination in accordance with the sample design.

Counts
The left testis and cauda epididymis from P1, P2, and Cohort 1A F1 males were weighed and then frozen at approximately -20C for subsequent determination of the number of homogenization-resistant spermatids and sperm per testis/cauda epididymis and per gram of testicular/epididymal tissue. The thawed testis or epididymis were minced, diluted, and stained with a fluorescent DNA-binding dye (HTM-IDENT, Hamilton-Thorne Research, Beverly, Massachusetts) and the spermatid or sperm count were determined from an aliquot loaded into the IVOS analyzer. Spermatid/sperm counts were evaluated in P1, P2, and Cohort 1A F1 males from the control and high-dose groups, as well as any males that failed to mate successfully during the mating period. Examination of epididymal sperm counts of the P1 control and high-dose males initially revealed a statistically significant decrease in the high-dose male sperm concentration per gram of epididymis but not total sperm count, thus triggering an analysis of the 300 and 1000 ppm dose groups.

Morphology
An aliquot of sperm suspension from P1, P2, and Cohort 1A F1 males were placed on a slide, and a smear prepared and air-dried for subsequent evaluation of sperm morphology. At least 200 sperm per male were evaluated and were classified as normal or abnormal as described by Filler (1993). Morphological evaluation of sperm was conducted using samples from the P1, P2, and Cohort 1A F1 males from the control and high-dose groups and any males that failed to mate successfully during the mating period. If treatment-related effects are observed, evaluation of sperm from the lower dose levels will be performed. Sperm morphology was scored blind with respect to treatment group.
Litter observations:
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Litter Data

Females were observed periodically for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day that one or more delivered fetuses were noted, and was designated as LD 0. The following information was collected and recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, and 21, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, and 21. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see Daily Observations). In addition, pup clinical observations were recorded on PND 0, 1, 4, 7, 14, and 21. Any pup found dead or sacrificed in moribund condition was sexed and examined grossly, to the extent possible, for external and visceral defects. These pups were preserved in neutral, phosphate-buffered 10% formalin. F2 litters were examined in the same manner as described above for F1 litters.

Anogenital Distance
Anogenital distance (absolute and relative to the cube root of body weight) was measured in all F1 pups on PND 1 (Gallavan et al., 1999). To the extent possible, the data collection order was counterbalanced by treatment group. Since a second generation was triggered, AGD was measured in F2 offspring as described above.

Culling and Culled Pups
To minimize variation in pup growth due to differences in litter size, all litters were standardized to ten pups per litter on PND 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were then randomly selected using a computer generated randomization procedure, so that five males and five females remained in each litter. If it was not possible to have five pups/sex in each litter, unequal numbers of males and females were retained (e.g., six males, four females). Litters with fewer than ten pups were not culled. Preferential culling of runts was not performed. Since a second generation was triggered, similar culling procedures were followed.

F1 and F2 Culled Pup Sampling for Thyroid Analysis
Pups were anesthetized on PND 4 using a mixture of isoflurane vapors and medical grade oxygen, and blood was collected via cardiac puncture and then transferred into serum separator tubes. To insure a sufficient volume for hormone analyses, blood was pooled by litter until a total of 10 samples/dose level were reached. Additional litters may have been used if there were insufficient pups (blood volume). Blood samples were processed as described below.
While under deep anesthesia, the culled pups were euthanized by decapitation. Following decapitation, all heads were removed and placed in 10% phosphate buffered formalin for histopathological examination of the thyroid gland.
All remaining culled pups from the litters were euthanized by decapitation while under deep isoflurane anesthesia or a sublingual administration of sodium pentobarbital solution, examined grossly and discarded. Since a second generation was bred, blood and tissue were collected from F2 culled pups.

Nipple/Areolae Retention
All offspring were evaluated for the presence of nipple/areolae on PND 12 in accordance with the methods described by McIntyre et al. (2001). The average number of nipples/areolae in male and female offspring in each litter was determined. The mean number of nipples/areolae for males and females in each dose level was calculated from these litter means. Observers were blind to treatment group when evaluating pups for the presence of nipples/areolae. There were no statistically or biologically significant, treatment-related increases in retained nipples/areolae in males on PND 12; thus Cohort 1A males were not examined for nipple/areolae retention at necropsy. Since a second generation was triggered, F2 offspring were evaluated for the presence of nipple/areolae on PND 12 as described above.

Weaning and Set Assignment
All litters were weaned on PND 21. If available, four male and four female F1 pups/litter were randomly selected and of these, one male and/or one female each were assigned to Cohort 1A, Cohort 1B, Cohort 2, or Cohort 3. The following prioritization plan was used (highest to lowest priority): Cohort 1A, Cohort 1B, Cohort 2A, Cohort 2B, and Cohort 3. One male and one female per litter were assigned to Cohort 1A. In addition, ten unselected control males if available were assigned to a splenic lymphocyte positive control group. If available, one male and one female per litter were assigned to Cohort 1B: the reproductive endpoints group (n > 20 males and 20 females/dose level). One male or one female per litter were assigned to Cohorts 2A (n = 11-12 males + 11 females/dose level) and 2B (n = 10 males + 10 females/dose level): the developmental neurotoxicity groups (with up to 22 litters represented in each Cohort) and Cohort 3 (n =10 males + 10 females): the developmental immunotoxicity group. In addition, five male and five female unselected control weanlings if available were assigned to an immunotoxicity positive control group. If there were insufficient litters from which to select both male and female offspring for groups 1, 2, or 3, additional animals were randomly selected from available litters as needed in order to obtain the required number of animals/dose level. Use of same sex littermates in Cohort 2A or 2B were avoided whenever possible. If there were insufficient pups to fill all of the designated pup assignments, pups were assigned in accordance with the prioritization plan outlined above.
Pups were implanted with transponders on ~ PND 17 for individual identification after weaning. Selected F1 offspring were housed in plastic cages with same sex littermates from PND 21-28, then housed individually thereafter.
All non-selected F1 and F2 weanlings were given a gross necropsy examination on PND 22, which included a gross pathologic assessment of reproductive organs. Blood was collected from a subset of weanlings (10/sex/dose) for thyroid hormone analyses. Body weight and organ weights (brain, spleen, liver, kidneys, and thymus) were collected from 10 pups/sex/dose (up to 20 litters represented, if possible) with preservation of these tissues plus mammary tissues for possible histopathological examination. Weanling necropsies are described in greater detail below.

Puberty Onset
All F1 animals (Cohorts 1-3) were observed daily for vaginal opening beginning on PND 26 (Cooper et al., 1989) or for balano-preputial separation beginning on PND 35 (Korenbrot et al., 1977). Age and body weight of the animals on the day these markers of puberty onset were recorded. Examination for puberty onset ceased upon acquisition, or on PND 43 (females) or 53 (males), whichever came first. Animals not acquiring these markers were assigned a value of 44 (females) or 54 (males) and designated as such in the appropriate tables. Any abnormalities of genital organs (e.g., persistent vaginal or preputial threads) were noted.
Postmortem examinations (parental animals):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

P1 Adults and Cohort 1A F1 Offspring

P1 males (fasted) were necropsied after ~18 weeks of exposure. Adult P1 females (fasted) were terminated on LD 22 after weaning of their litters, or at least 24 days after the end of the mating period for females not producing a litter. F1 male and female adults (fasted) from Cohort 1A were submitted for necropsy on approximately PND 90 (± 1 day). Animals were weighed in the animal room and vaginal lavage smears were prepared from all surviving P1 and Cohort 1A F1 females prior to transportation to the necropsy room. The animals were anesthetized by the inhalation of a mixture of isoflurane vapors and medical grade oxygen, and blood was collected from the orbital sinus for 10/sex/dose for clinical pathology and thyroid hormone analysis (both P1 and Cohort 1A animals). The animals were then placed in a CO2 chamber to continue anesthesia and euthanasia. A complete necropsy was conducted on all animals by a veterinary pathologist or a trained examiner, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.
The uteri of all P1 females were stained with an aqueous solution of 10% sodium sulfide stain after removal of the ovaries (Kopf et al., 1964) and were examined for the presence and number of implantation sites. The uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, brain, pituitary (weighed after fixation), liver, kidneys, heart, thymus, adrenal glands, spleen, submandibular and mesenteric lymph nodes (weighed after fixation in 10/sex/dose Cohort 1A F1 only) and thyroid with parathyroid glands (weighed after fixation) were recorded, and the organ:body weight ratios calculated. In addition, weights of the left testis and left cauda epididymis were collected in P1 males and Cohort 1A F1 males. These data were used to calculate sperm counts (cauda epididymis) and spermatid counts (testis). Since there was a statistically-identified change on prostate weights, the dorsolateral and ventral segments were dissected after fixation and weighed separately for the Cohort 1A F1 males in accordance to the guideline (OECD 443).
Representative samples of tissues were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the right testis, right epididymis, left caput and corpus epididymides, and ovaries were preserved in Bouin’s fixative. Transponders were removed and placed in formalin jars with the tissues.
For the splenic subpopulation analysis positive control group, animals were weighed in the animal room and necropsied as above with the exceptions that only the spleen and thymus were trimmed and weighed, organ:body weight ratios calculated, and preserved in neutral, phosphate buffered 10% formalin. No other necropsy procedures were conducted on the positive control group.
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy was necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Dams unable to deliver or exhibiting signs of severe dystocia were humanely euthanized and necropsied. Similar necropsy procedures were followed for animals found dead or euthanized prior to the scheduled necropsy except that terminal body and organ weights were not recorded and the testes, epididymides and ovaries were preserved in neutral, phosphate-buffered 10% formalin. A complete set of tissues was saved from these animals.

Sperm Analysis – P1 and P2 Adults and Cohort 1A F1 Males

Sperm parameters were evaluated in P1, P2, and Cohort 1A F1 males at termination. Unless circumstances dictate otherwise, the left and right epididymides and testes were allocated as follows: right epididymis – motility and histopathology; left epididymis – counts; right testis – histopathology; left; testis – counts.

Motility
Sperm motility was evaluated in P1, P2, and Cohort F1 males from all dose groups. Immediately after euthanasia of males and isolation of their epididymides, a small sample of sperm from the right cauda epididymis was expressed into a dish containing pre-warmed Modified Sperm Washing Medium (Irvine Scientific, Santa Ana, California) and was incubated for approximately 2-3 minutes. An aliquot of the incubated sperm suspension was placed on a slide and loaded into the Hamilton-Thorne Integrated Visual Optical System (IVOS; Hamilton-Thorne Research, Beverly, Massachusetts) for the determination of total percent motile (showing any motion) and percent progressively motile (showing net forward motion) sperm. After sperm were released, the epididymis was placed in Bouin’s fixative and subjected to histopathologic examination in accordance with the sample design (see below).

Counts
The left testis and cauda epididymis from P1, P2, and Cohort 1A F1 males were weighed and then frozen at approximately -20C for subsequent determination of the number of homogenization-resistant spermatids and sperm per testis/cauda epididymis and per gram of testicular/epididymal tissue. The thawed testis or epididymis were minced, diluted, and stained with a fluorescent DNA-binding dye (HTM-IDENT, Hamilton-Thorne Research, Beverly, Massachusetts) and the spermatid or sperm count were determined from an aliquot loaded into the IVOS analyzer. Spermatid/sperm counts were evaluated in P1, P2, and Cohort 1A F1 males from the control and high-dose groups, as well as any males that failed to mate successfully during the mating period. Examination of epididymal sperm counts of the P1 control and high-dose males initially revealed a statistically significant decrease in the high-dose male sperm concentration per gram of epididymis but not total sperm count, thus triggering an analysis of the 300 and 1000 ppm dose groups.

Morphology
An aliquot of sperm suspension from P1, P2, and Cohort 1A F1 males were placed on a slide, and a smear prepared and air-dried for subsequent evaluation of sperm morphology. At least 200 sperm per male were evaluated and were classified as normal or abnormal as described by Filler (1993). Morphological evaluation of sperm was conducted using samples from the P1, P2, and Cohort 1A F1 males from the control and high-dose groups and any males that failed to mate successfully during the mating period. If treatment-related effects are observed, evaluation of sperm from the lower dose levels will be performed. Sperm morphology was scored blind with respect to treatment group.

Histopathology – P1 Adults and Cohort 1A F1 Offspring
Histopathological examination of the tissues were conducted on the control and high-dose groups of P1 adults and Cohort 1A F1 offspring. Examination of tissues from the remaining groups were limited to those tissues that demonstrated treatment-related histopathologic effects at the high dose livers (males and females), kidneys (males and females), and urinary bladder (males), relevant gross lesions, and reproductive organs from animals that failed to mate or exhibit reduced fertility (P1 adults). Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope.
Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of the testis from both control and high-dose males was embedded in paraffin, sectioned at 5 µm and stained with modified periodic acid Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis were qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, the testis was examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). The sections of testis included rete testis which were examined for all Cohort 1A F1 males. Examination of the epididymis included the caput, corpus and cauda. The vas deferens were also examined.
Examination of the ovaries from P1 control and high-dose females included a qualitative assessment of follicle stages with an emphasis on the possible depletion of primordial follicles. For Cohort 1A F1 females, examination of the ovaries included enumeration of primordial follicles and corpora lutea using a method similar to Bucci et al. (1997) with additional recommendations from Heindel (1999) and Bolon et al. (1997). All Cohort 1A F1 females in the control and high-dose groups terminated at scheduled necropsy were selected for this examination. The examination was conducted with the observer blinded to treatment group. The Cohort 1A F1 ovarian follicle and corpora lutea counts were conducted as follows:
1. A quantitative evaluation of the Cohort 1A primordial and growing follicles as well as corpora lutea was conducted in at least 1% of the ovary in control and high-dose females as a screening measure for potential adverse effects. No adverse effects were observed on follicle or corpora lutea counts, therefore, no further action was taken.
Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change would neither be expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate would not have been life-threatening. A severe grade was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may have been life-threatening.
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. A complete set of tissues were examined from rats found dead or moribund (including animals with dystocia). Histopathological examination was conducted in a similar manner as described above, except that the testes were stained with hematoxylin and eosin. Similar necropsy procedures were followed for animals found dead or euthanized prior to the scheduled necropsy except that terminal body and organ weights were not recorded. There was no assessment of sperm parameters or quantitative ovarian follicle counts on dead or moribund animals.

Thyroid Histopathology – F1 PND 4 Culled Pups
Histopathological examination of the thyroid gland from one randomly selected control and high-dose F1 PND 4 culled pup from litters that had been sampled for thyroid hormone analysis was conducted to assist with interpretation of the thyroid hormone data. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope.

Anatomic Pathology – Cohort 1B P2 Animals
Since a second mating was required, Cohort 1B P2 males were submitted for necropsy two weeks after the end of mating and Cohort 1B P2 females were necropsied at the end of lactation. Animals were weighed in the animal room and vaginal lavage smears were prepared from all surviving Cohort 1B P2 females prior to transportation to the necropsy room. The animals were anesthetized by the inhalation of a mixture of isoflurane vapors and medical grade oxygen and blood was collected from the orbital sinus for 10/sex/dose for clinical pathology and thyroid hormone analysis. The animals were then placed in a CO2 chamber to continue anesthesia and sacrifice.

A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.
Since Cohort 1B P2 females were bred, the uteri were stained with an aqueous solution of 10% sodium sulfide stain after removal of the ovaries (Kopf et al., 1964) and were examined for the presence and number of implantation sites. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, pituitary (weighed after fixation), liver, and kidneys were recorded, and the organ:body weight ratios calculated for Cohort 1B P2 animals.
Representative samples of tissues listed above, plus vagina, were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the testes, epididymides, and ovaries were preserved in Bouin’s fixative. During the course of the scheduled necropsies, a decision was made to collect additional tissues such as the urinary bladder and stomach because of the possibility of these tissues being potential target organs based on gross observations. Thus, in addition to the tissues collected, the urinary bladder from the remaining Cohort 1B P2 males necropsied on October 25-26, 2017 and all Cohort 1B P2 females, and the stomach from the remaining Cohort 1B P2 females necropsied on November 08-18, 2017 were collected and preserved in neutral phosphate-buffered 10% formalin. Transponders were removed and placed in formalin jars with the tissues. Liver, kidneys, and urinary bladders of the Cohort 1B P2 animals (males and females) were processed to block stage and taken to slide since these organs had increased weights and/or were identified as target organs. In addition, all relevant gross lesions were processed to block stage and taken to slide.
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Cohort 1B P2 dams unable to deliver or exhibiting signs of severe dystocia were humanely euthanized and necropsied. Similar necropsy procedures were followed for animals found dead or euthanized prior to the scheduled necropsy except that terminal body and organ weights were not recorded.

Histopathology – Cohort 1B P2 Animals
Histopathological examination of the liver, kidneys, and urinary bladder were conducted on the control and high-dose groups of P2 animals since these tissues had increased weights and/or were identified as target organs in the P1 animals. Examination of tissues from the remaining groups were limited to those tissues that demonstrated treatment-related histopathologic effects at the high dose (liver, kidneys, and urinary bladder). In addition, all relevant gross lesions were also microscopically examined. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope.

Thyroid Histopathology – F2 PND 4 Culled Pups
Histopathological examination of the thyroid gland from one randomly selected control and high-dose F2 PND 4 culled pup from litters that had been sampled for thyroid hormone analysis was conducted to assist with interpretation of the thyroid hormone data. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope.

Anatomic Pathology – Cohort 2A F1 Offspring
Cohort 2A F1 males and females (fasted) were submitted for necropsy on PND 78. Animals were weighed in the animal room prior to transportation to the necropsy room. The Cohort 2A F1 animals underwent perfusion for fixation of nervous system tissues for neuropathology (described below).

Neurotoxicity Group – Cohort 2A F1 Offspring

Necropsy
All animals in Cohort 2A surviving to the scheduled necropsy (PND 78) for perfusion were counterbalanced and given an intraperitoneal injection of heparin (1.0 ml for male and 0.6 ml for female rats) approximately 10 minutes prior to perfusion. This dose was chosen based on the upper limit of the body weight ranges for this strain, age, and sex of rats and was calculated based on an approximate dose of 0.2 ml heparin (10,000 USP/ml) per 100 grams body weight. Animals were anesthetized with O2/Isoflurane. While under deep anesthesia the heart was exposed, the left ventricle cannulated, and the right atrium was incised.

Animals were perfused by gravity pressure with 0.05 M phosphate buffer containing sodium nitrite followed by a phosphate-buffered solution of 1.5% glutaraldehyde - 4% formaldehyde (c 540 mOs).
Tissues were examined for gross pathologic alterations by a veterinary pathologist assisted by a team of trained individuals. The brain (excluding the olfactory lobes), head, spinal column with spinal cord, fore- and hindlimbs, and tail were trimmed to remove excessive skin and muscle; muscles from the hindlimbs were reflected to further expose the nerves. All tissues were immersed in the glutaraldehyde/formaldehyde fixative. Additional tissues (liver, kidneys, urinary bladder, and gross lesions) were saved in glutaraldehyde/formaldehyde for potential histopathological examination.

Histopathology
Tissues for neuropathologic evaluation were prepared from all animals in the control and high-dose groups. In addition, appropriate brain sections from the low- and intermediate-dose groups were processed to slide stage for possible morphometric evaluation. Nine cross-sections of the brain were prepared using a hand-held, single-edged industrial blade from the following structures: olfactory bulb, cerebrum (frontal, parietal, temporal and occipital lobes, including the hippocampus and basal ganglia), thalamus/hypothalamus, midbrain, pons, medulla oblongata, and cerebellum. For morphometric purposes, two transverse tissue blocks were cut through the cerebrum and midbrain (block #3, and #4). A longitudinal (anterior to posterior) cut was made midway through the cerebellum after it was removed from the midbrain (block #10). The following gross rostral landmarks for each block were used: block #3 – optic chiasm, block #4 – anterior edge of infundibulum, and block #10 dorsal midline apex of the cerebellum. Blocks #3 and #4 contained the following structures: cerebrum (frontal and parietal lobes), thalamus/hypothalamus, and midbrain. These tissues were processed by standard histologic procedures, embedded in paraffin, sectioned approximately 6-µm thick and stained with hematoxylin and eosin and coverslipped. Sections of the brain and spinal cord were also stained with Fluoro-Jade according to Schmued et al. (1997). A sufficient number of sections were cut from each block to meet the criteria of finding the appropriate microscopic landmarks for the purpose of brain measurement. Microscopic landmarks were as follows: block #3 – greatest thickness at the midpoint of the anterior commissure, block #4 – contact/close proximity of the dentate gyrus and the polymorph layer of the dentate gyrus, and block #10 – midline longitudinal section that contained the medial cerebellar nucleus. The most appropriate section from each block was used for the microscopic measurements.
In addition, sections were prepared from the trigeminal ganglion and nerve, pituitary gland, eyes (retina) with optic nerves, spinal cord (cervical and lumbar), olfactory epithelium, and skeletal muscles (gastrocnemius and anterior tibial). These tissues were processed by standard histologic procedures, embedded in paraffin, sectioned approximately 6-µm thick and stained with hematoxylin and eosin and coverslipped.
Spinal nerve roots (cervical and lumbar), dorsal root ganglia (cervical and lumbar), and peripheral nerves (sciatic, tibial (proximal and distal (muscular) - at the knee and calf muscle branches) and sural) were osmicated, embedded in epoxy resin, sectioned approximately 2 to 3 µm thick and stained with toluidine blue.
Tissues were evaluated by a veterinary pathologist using a light microscope. Histopathological findings were subjectively graded as appropriate to assess the potential effects of treatment with regard to the contribution of a specific lesion to the health status of an animal. A grade of very slight was used for conditions present in excess of the normal textbook appearance of an organ/tissue, but were of minimal severity and were not expected to significantly affect the function of the specific organ/tissue involved nor have a significant effect on the overall health of the animal. Lesions of this severity involved only a minor portion of the affected organ. Categories of slight, moderate, severe or very severe were available for potential use for lesions of greater severity.

Brain Weight and Gross Measurements
Brain weight and gross measurements were recorded on all dose groups surviving to the scheduled necropsy. Brains (excluding the olfactory lobes) were weighed following 7-15 days of fixation. Linear measurements consisted of the: 1) cerebral length (L2 – anterior to posterior, excluding olfactory lobes) and width (L3 – maximum), and 2) cerebellar length (L10 – anterior to posterior) and width (L5 – maximum), which were obtained using a hand-held, electronic digital slide caliper, whose calibration was checked prior to use each day.

Microscopic Brain Measurements
Microscopic brain measurements were recorded on all animals in the control and high-dose groups surviving to the scheduled necropsy. Microscopic brain measurements were obtained from a number of anatomical structures in tissue sections stained with H&E from blocks #3, 4, and 10 having the appropriate landmarks. Images of each section of brain were digitally captured using Aperio Versa 8 Leica Imaging system were saved and managed in eSlideManager version (version 12.3.2.5030). Simple morphometric measurements were obtained using the image analysis software Halo (v2.0.1145.14) to obtain distance measurements as follows: 1) distance measurement - created a measurement of the distance between two user-selected features. Measurements were excluded or altered (e.g., combination of multiple smaller measurements as necessary) due to possible microscopic artifacts, such as missing portion(s) of tissue, tears in tissue etc. Morphometric data obtained using Halo were electronically transferred to Microsoft Excel spreadsheets.

Anatomic Pathology – Cohort 3 F1 Offspring

Cohort 3 F1 males and females including the positive control animals (fasted) were submitted for necropsy on PND 56 ± 3. Animals were weighed in the animal room prior to transportation to the necropsy room. The animals were anesthetized with a mixture of isoflurane vapors and medical oxygen, and blood samples obtained from the orbital sinus.

Necropsy
Following blood collection, the Cohort 3 animals were placed in a CO2 chamber to continue anesthesia and euthanasia. A complete necropsy was conducted on all animals (excluding IPC group) by a veterinary pathologist or a trained gross examiner, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. Weights of the spleen and thymus were recorded and organ:terminal body weight ratios calculated. Representative samples of spleen, thymus and gross lesions were collected and preserved in neutral, phosphate buffered 10% formalin for possible histopathological evaluation.
For the IPC group, only the spleen and thymus were trimmed and weighed, organ:body weight ratios calculated, and preserved in neutral, phosphate buffered 10% formalin. No other necropsy procedures were conducted on the IPC group (with the exception of blood collection for anti-SRBC analysis).
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were followed for animals found dead or euthanized prior to the scheduled necropsy except that terminal body and organ weights were not recorded and blood was not collected.
Postmortem examinations (offspring):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

P1 Adults and Cohort 1A F1 Offspring

P1 males (fasted) were necropsied after ~18 weeks of exposure. Adult P1 females (fasted) were terminated on LD 22 after weaning of their litters, or at least 24 days after the end of the mating period for females not producing a litter. F1 male and female adults (fasted) from Cohort 1A were submitted for necropsy on approximately PND 90 (± 1 day). Animals were weighed in the animal room and vaginal lavage smears were prepared from all surviving P1 and Cohort 1A F1 females prior to transportation to the necropsy room. The animals were anesthetized by the inhalation of a mixture of isoflurane vapors and medical grade oxygen, and blood was collected from the orbital sinus for 10/sex/dose for clinical pathology and thyroid hormone analysis (both P1 and Cohort 1A animals). The animals were then placed in a CO2 chamber to continue anesthesia and sacrifice.
A complete necropsy was conducted on all animals by a veterinary pathologist or a trained examiner, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.
The uteri of all P1 females were stained with an aqueous solution of 10% sodium sulfide stain after removal of the ovaries (Kopf et al., 1964) and were examined for the presence and number of implantation sites. The uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, brain, pituitary (weighed after fixation), liver, kidneys, heart, thymus, adrenal glands, spleen, submandibular and mesenteric lymph nodes (weighed after fixation in 10/sex/dose Cohort 1A F1 only) and thyroid with parathyroid glands (weighed after fixation) were recorded, and the organ:body weight ratios calculated. In addition, weights of the left testis and left cauda epididymis were collected in P1 males and Cohort 1A F1 males. These data were used to calculate sperm counts (cauda epididymis) and spermatid counts (testis). Since there was a statistically-identified change on prostate weights, the dorsolateral and ventral segments were dissected after fixation and weighed separately for the Cohort 1A F1 males in accordance to the guideline (OECD 443).
Representative samples of tissues were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the right testis, right epididymis, left caput and corpus epididymides, and ovaries were preserved in Bouin’s fixative. Transponders were removed and placed in formalin jars with the tissues.
For the splenic subpopulation analysis positive control group, animals were weighed in the animal room and necropsied as above with the exceptions that only the spleen and thymus were trimmed and weighed, organ:body weight ratios calculated, and preserved in neutral, phosphate buffered 10% formalin. No other necropsy procedures were conducted on the positive control group.
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy was necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Dams unable to deliver or exhibiting signs of severe dystocia were humanely euthanized and necropsied. Similar necropsy procedures were followed for animals found dead or euthanized prior to the scheduled necropsy except that terminal body and organ weights were not recorded and the testes, epididymides and ovaries were preserved in neutral, phosphate-buffered 10% formalin. A complete set of tissues was saved from these animals.

Sperm Analysis – P1 and P2 Adults and Cohort 1A F1 Males

Sperm parameters were evaluated in P1, P2, and Cohort 1A F1 males at termination. Unless circumstances dictate otherwise, the left and right epididymides and testes were allocated as follows: right epididymis – motility and histopathology; left epididymis – counts; right testis – histopathology; left; testis – counts.

Motility
Sperm motility was evaluated in P1, P2, and Cohort F1 males from all dose groups. Immediately after euthanasia of males and isolation of their epididymides, a small sample of sperm from the right cauda epididymis was expressed into a dish containing pre-warmed Modified Sperm Washing Medium (Irvine Scientific, Santa Ana, California) and was incubated for approximately 2-3 minutes. An aliquot of the incubated sperm suspension was placed on a slide and loaded into the Hamilton-Thorne Integrated Visual Optical System (IVOS; Hamilton-Thorne Research, Beverly, Massachusetts) for the determination of total percent motile (showing any motion) and percent progressively motile (showing net forward motion) sperm. After sperm were released, the epididymis was placed in Bouin’s fixative and subjected to histopathologic examination in accordance with the sample design (see below).

Counts
The left testis and cauda epididymis from P1, P2, and Cohort 1A F1 males were weighed and then frozen at approximately -20C for subsequent determination of the number of homogenization-resistant spermatids and sperm per testis/cauda epididymis and per gram of testicular/epididymal tissue. The thawed testis or epididymis were minced, diluted, and stained with a fluorescent DNA-binding dye (HTM-IDENT, Hamilton-Thorne Research, Beverly, Massachusetts) and the spermatid or sperm count were determined from an aliquot loaded into the IVOS analyzer. Spermatid/sperm counts were evaluated in P1, P2, and Cohort 1A F1 males from the control and high-dose groups, as well as any males that failed to mate successfully during the mating period. Examination of epididymal sperm counts of the P1 control and high-dose males initially revealed a statistically significant decrease in the high-dose male sperm concentration per gram of epididymis but not total sperm count, thus triggering an analysis of the 300 and 1000 ppm dose groups.

Morphology
An aliquot of sperm suspension from P1, P2, and Cohort 1A F1 males were placed on a slide, and a smear prepared and air-dried for subsequent evaluation of sperm morphology. At least 200 sperm per male were evaluated and were classified as normal or abnormal as described by Filler (1993). Morphological evaluation of sperm was conducted using samples from the P1, P2, and Cohort 1A F1 males from the control and high-dose groups and any males that failed to mate successfully during the mating period. If treatment-related effects are observed, evaluation of sperm from the lower dose levels will be performed. Sperm morphology was scored blind with respect to treatment group.

Histopathology – P1 Adults and Cohort 1A F1 Offspring
Histopathological examination of the tissues were conducted on the control and high-dose groups of P1 adults and Cohort 1A F1 offspring. Examination of tissues from the remaining groups were limited to those tissues that demonstrated treatment-related histopathologic effects at the high dose livers (males and females), kidneys (males and females), and urinary bladder (males), relevant gross lesions, and reproductive organs from animals that failed to mate or exhibit reduced fertility (P1 adults). Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope.
Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of the testis from both control and high-dose males was embedded in paraffin, sectioned at 5 µm and stained with modified periodic acid Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis were qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, the testis was examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). The sections of testis included rete testis which were examined for all Cohort 1A F1 males. Examination of the epididymis included the caput, corpus and cauda. The vas deferens were also examined.
Examination of the ovaries from P1 control and high-dose females included a qualitative assessment of follicle stages with an emphasis on the possible depletion of primordial follicles. For Cohort 1A F1 females, examination of the ovaries included enumeration of primordial follicles and corpora lutea using a method similar to Bucci et al. (1997) with additional recommendations from Heindel (1999) and Bolon et al. (1997). All Cohort 1A F1 females in the control and high-dose groups terminated at scheduled necropsy were selected for this examination. The examination was conducted with the observer blinded to treatment group. The Cohort 1A F1 ovarian follicle and corpora lutea counts were conducted as follows:
1. A quantitative evaluation of the Cohort 1A primordial and growing follicles as well as corpora lutea was conducted in at least 1% of the ovary in control and high-dose females as a screening measure for potential adverse effects. No adverse effects were observed on follicle or corpora lutea counts, therefore, no further action was taken.
Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change would neither be expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate would not have been life-threatening. A severe grade was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may have been life-threatening.
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. A complete set of tissues were examined from rats found dead or moribund (including animals with dystocia). Histopathological examination was conducted in a similar manner as described above, except that the testes were stained with hematoxylin and eosin. Similar necropsy procedures were followed for animals found dead or euthanized prior to the scheduled necropsy except that terminal body and organ weights were not recorded. There was no assessment of sperm parameters or quantitative ovarian follicle counts on dead or moribund animals.

Thyroid Histopathology – F1 PND 4 Culled Pups
Histopathological examination of the thyroid gland from one randomly selected control and high-dose F1 PND 4 culled pup from litters that had been sampled for thyroid hormone analysis was conducted to assist with interpretation of the thyroid hormone data. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope.

Anatomic Pathology – Cohort 1B P2 Animals
Since a second mating was required, Cohort 1B P2 males were submitted for necropsy two weeks after the end of mating and Cohort 1B P2 females were necropsied at the end of lactation. Animals were weighed in the animal room and vaginal lavage smears were prepared from all surviving Cohort 1B P2 females prior to transportation to the necropsy room. The animals were anesthetized by the inhalation of a mixture of isoflurane vapors and medical grade oxygen and blood was collected from the orbital sinus for 10/sex/dose for clinical pathology and thyroid hormone analysis. The animals were then placed in a CO2 chamber to continue anesthesia and euthanasia.
A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.
Since Cohort 1B P2 females were bred, the uteri were stained with an aqueous solution of 10% sodium sulfide stain after removal of the ovaries (Kopf et al., 1964) and were examined for the presence and number of implantation sites. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, pituitary (weighed after fixation), liver, and kidneys were recorded, and the organ:body weight ratios calculated for Cohort 1B P2 animals.
Representative samples of tissues listed above, plus vagina were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the testes, epididymides, and ovaries were preserved in Bouin’s fixative. During the course of the scheduled necropsies, a decision was made to collect additional tissues such as the urinary bladder and stomach because of the possibility of these tissues being potential target organs based on gross observations. Thus, in addition to the tissues, the urinary bladder from the remaining Cohort 1B P2 males necropsied on October 25-26, 2017 and all Cohort 1B P2 females, and the stomach from the remaining Cohort 1B P2 females necropsied on November 08-18, 2017 were collected and preserved in neutral phosphate-buffered 10% formalin. Transponders were removed and placed in formalin jars with the tissues. Liver, kidneys, and urinary bladders of the Cohort 1B P2 animals (males and females) were processed to block stage and taken to slide since these organs had increased weights and/or were identified as target organs. In addition, all relevant gross lesions were processed to block stage and taken to slide.
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Cohort 1B P2 dams unable to deliver or exhibiting signs of severe dystocia were humanely euthanized and necropsied. Similar necropsy procedures were followed for animals found dead or euthanized prior to the scheduled necropsy except that terminal body and organ weights were not recorded.

Histopathology – Cohort 1B P2 Animals
Histopathological examination of the liver, kidneys, and urinary bladder were conducted on the control and high-dose groups of P2 animals since these tissues had increased weights and/or were identified as target organs in the P1 animals. Examination of tissues from the remaining groups were limited to those tissues that demonstrated treatment-related histopathologic effects at the high dose (liver, kidneys, and urinary bladder). In addition, all relevant gross lesions were also microscopically examined. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope.

Thyroid Histopathology – F2 PND 4 Culled Pups
Histopathological examination of the thyroid gland from one randomly selected control and high-dose F2 PND 4 culled pup from litters that had been sampled for thyroid hormone analysis was conducted to assist with interpretation of the thyroid hormone data. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope.

Anatomic Pathology – Cohort 2A F1 Offspring
Cohort 2A F1 males and females (fasted) were submitted for necropsy on PND 78. Animals were weighed in the animal room prior to transportation to the necropsy room. The Cohort 2A F1 animals underwent perfusion for fixation of nervous system tissues for neuropathology (described below).

Neurotoxicity Group – Cohort 2A F1 Offspring

Necropsy
All animals in Cohort 2A surviving to the scheduled necropsy (PND 78) for perfusion were counterbalanced and given an intraperitoneal injection of heparin (1.0 ml for male and 0.6 ml for female rats) approximately 10 minutes prior to perfusion. This dose was chosen based on the upper limit of the body weight ranges for this strain, age, and sex of rats and was calculated based on an approximate dose of 0.2 ml heparin (10,000 USP/ml) per 100 grams body weight. Animals were anesthetized with O2/Isoflurane. While under deep anesthesia the heart was exposed, the left ventricle cannulated, and the right atrium was incised.

Animals were perfused by gravity pressure with 0.05 M phosphate buffer containing sodium nitrite followed by a phosphate-buffered solution of 1.5% glutaraldehyde - 4% formaldehyde (c 540 mOs).
Tissues were examined for gross pathologic alterations by a veterinary pathologist assisted by a team of trained individuals. The brain (excluding the olfactory lobes), head, spinal column with spinal cord, fore- and hindlimbs, and tail were trimmed to remove excessive skin and muscle; muscles from the hindlimbs were reflected to further expose the nerves. All tissues were immersed in the glutaraldehyde/formaldehyde fixative. Additional tissues (liver, kidneys, urinary bladder, and gross lesions) were saved in glutaraldehyde/formaldehyde for potential histopathological examination.

Histopathology
Tissues for neuropathologic evaluation were prepared from all animals in the control and high-dose groups. In addition, appropriate brain sections from the low- and intermediate-dose groups were processed to slide stage for possible morphometric evaluation. Nine cross-sections of the brain were prepared using a hand-held, single-edged industrial blade from the following structures: olfactory bulb, cerebrum (frontal, parietal, temporal and occipital lobes, including the hippocampus and basal ganglia), thalamus/hypothalamus, midbrain, pons, medulla oblongata, and cerebellum. For morphometric purposes, two transverse tissue blocks were cut through the cerebrum and midbrain (block #3, and #4). A longitudinal (anterior to posterior) cut was made midway through the cerebellum after it was removed from the midbrain (block #10). The following gross rostral landmarks for each block were used: block #3 – optic chiasm, block #4 – anterior edge of infundibulum, and block #10 dorsal midline apex of the cerebellum. Blocks #3 and #4 contained the following structures: cerebrum (frontal and parietal lobes), thalamus/hypothalamus, and midbrain. These tissues were processed by standard histologic procedures, embedded in paraffin, sectioned approximately 6-µm thick and stained with hematoxylin and eosin and coverslipped. Sections of the brain and spinal cord were also stained with Fluoro-Jade according to Schmued et al. (1997). A sufficient number of sections were cut from each block to meet the criteria of finding the appropriate microscopic landmarks for the purpose of brain measurement. Microscopic landmarks were as follows: block #3 – greatest thickness at the midpoint of the anterior commissure, block #4 – contact/close proximity of the dentate gyrus and the polymorph layer of the dentate gyrus, and block #10 – midline longitudinal section that contained the medial cerebellar nucleus. The most appropriate section from each block was used for the microscopic measurements.
In addition, sections were prepared from the trigeminal ganglion and nerve, pituitary gland, eyes (retina) with optic nerves, spinal cord (cervical and lumbar), olfactory epithelium, and skeletal muscles (gastrocnemius and anterior tibial). These tissues were processed by standard histologic procedures, embedded in paraffin, sectioned approximately 6-µm thick and stained with hematoxylin and eosin and coverslipped.
Spinal nerve roots (cervical and lumbar), dorsal root ganglia (cervical and lumbar), and peripheral nerves (sciatic, tibial (proximal and distal (muscular) - at the knee and calf muscle branches) and sural) were osmicated, embedded in epoxy resin, sectioned approximately 2 to 3 µm thick and stained with toluidine blue.
Tissues were evaluated by a veterinary pathologist using a light microscope. Histopathological findings were subjectively graded as appropriate to assess the potential effects of treatment with regard to the contribution of a specific lesion to the health status of an animal. A grade of very slight was used for conditions present in excess of the normal textbook appearance of an organ/tissue, but were of minimal severity and were not expected to significantly affect the function of the specific organ/tissue involved nor have a significant effect on the overall health of the animal. Lesions of this severity involved only a minor portion of the affected organ. Categories of slight, moderate, severe or very severe were available for potential use for lesions of greater severity.

Brain Weight and Gross Measurements
Brain weight and gross measurements were recorded on all dose groups surviving to the scheduled necropsy. Brains (excluding the olfactory lobes) were weighed following 7-15 days of fixation. Linear measurements consisted of the: 1) cerebral length (L2 – anterior to posterior, excluding olfactory lobes) and width (L3 – maximum), and 2) cerebellar length (L10 – anterior to posterior) and width (L5 – maximum), which were obtained using a hand-held, electronic digital slide caliper, whose calibration was checked prior to use each day.

Microscopic Brain Measurements
Microscopic brain measurements were recorded on all animals in the control and high-dose groups surviving to the scheduled necropsy. Microscopic brain measurements were obtained from a number of anatomical structures in tissue sections stained with H&E from blocks #3, 4, and 10 having the appropriate landmarks. Images of each section of brain were digitally captured using Aperio Versa 8 Leica Imaging system were saved and managed in eSlideManager version (version 12.3.2.5030). Simple morphometric measurements were obtained using the image analysis software Halo (v2.0.1145.14) to obtain distance measurements as follows: 1) distance measurement - created a measurement of the distance between two user-selected features. Measurements were excluded or altered (e.g., combination of multiple smaller measurements as necessary) due to possible microscopic artifacts, such as missing portion(s) of tissue, tears in tissue etc. Morphometric data obtained using Halo were electronically transferred to Microsoft Excel spreadsheets.

Anatomic Pathology – Cohort 3 F1 Offspring

Cohort 3 F1 males and females including the positive control animals (fasted) were submitted for necropsy on PND 56 ± 3. Animals were weighed in the animal room prior to transportation to the necropsy room. The animals were anesthetized with a mixture of isoflurane vapors and medical oxygen, and blood samples obtained from the orbital sinus.

Necropsy
Following blood collection, the Cohort 3 animals were placed in a CO2 chamber to continue anesthesia and sacrifice. A complete necropsy was conducted on all animals (excluding IPC group) by a veterinary pathologist or a trained gross examiner, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. Weights of the spleen and thymus were recorded and organ:terminal body weight ratios calculated. Representative samples of spleen, thymus and gross lesions were collected and preserved in neutral, phosphate buffered 10% formalin for possible histopathological evaluation.
For the IPC group, only the spleen and thymus were trimmed and weighed, organ:body weight ratios calculated, and preserved in neutral, phosphate buffered 10% formalin. No other necropsy procedures were conducted on the IPC group (with the exception of blood collection for anti-SRBC analysis).
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were followed for animals found dead or euthanized prior to the scheduled necropsy except that terminal body and organ weights were not recorded and blood was not collected.
Statistics:
Statistics are included in "other information" due to the restriction on a reasonable number of characters in this field box
Reproductive indices:
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Litter Data
Females were observed periodically for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day that one or more delivered fetuses were noted, and was designated as LD 0. The following information was collected and recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, and 21, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, and 21. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see Daily Observations). In addition, pup clinical observations were recorded on PND 0, 1, 4, 7, 14, and 21. Any pup found dead or sacrificed in moribund condition was sexed and examined grossly, to the extent possible, for external and visceral defects. These pups were preserved in neutral, phosphate-buffered 10% formalin. F2 litters were examined in the same manner as described above for F1 litters.
Offspring viability indices:
The following information was collected and recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, and 21, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, and 21.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Examinations performed on all animals prior to the study start revealed that all animals were in good health. P1 males and females, and satellite females did not exhibit any treatment-related clinical observations throughout the term of the study. Clinical observations recorded for P1 animals during the study were isolated occurrences and/or deemed to be spontaneous occurrences common to this strain/age of rat and unrelated to exposure. One mid-dose male (animal 949) was sent to necropsy for animal welfare concerns associated with a probable nasal fracture. The animal exhibited the following clinical signs: red periocular and perinasal soiling, maloccluded incisors and trauma to its muzzle. During the gestation phase, one low dose dame (dam 1021) displayed vulvar discharge on GD 14, which subsequently resolved by the next observation on GD 17. This isolated observation was deemed unrelated to treatment and this dam delivered a litter with 16 live pups on GD 21, with no further clinical observations. During the lactation phase of the study, two low-dose dams (dam 1028 and 1030) and two mid-dose dams (dam 1049 and 1069) had dystocia (difficult birth). Dam 1028 given 300 ppm delivered 13 live pups on GD 22 (LD 0), but then appeared pale, had blood in the cage and delivered one dead pup on LD 2. Dam 1030 given 300 ppm delivered 13 live and 3 dead pups on GD 22 (LD 0), but then appeared pale, had labored respiration, an ungroomed appearance, a red vulvar discharge and decreased activity on LD 1 and was sent in a moribund condition to necropsy on TD 99 (LD 1). At necropsy, dam 1030 had cecal edema and hemorrhages, hemolyzed blood and ascites within the abdominal cavity, dark kidneys, a pale liver and lungs, stomach ulcers and the uterus contained two dead fetuses and retained placentas. Dam 1049 given 1000 ppm delivered 7 live and 2 dead pups on GD 22 (LD 0) but then appeared pale, had a red vulvar discharge, and delivered a dead and subsequently cannibalized pup on LD 1. Dam 1069 given 1000 ppm delivered 12 live pups on GD 22 (LD 0) but then appeared pale, had a red vulvar discharge and had three cannibalized pups in the cage on LD 1. There was no evidence of dystocia in the high-dose dams. Aside from dam #1030, dams exhibiting dystocia survived to scheduled necropsy. Although there was an increased incidence of dystocia observed in the low- and mid-dose dams, this observation was deemed unrelated to treatment because 1) there was no incidence of dystocia in the high-dose dams and
2) breeding a second generation was triggered based on the incidences of dystocia observed in the P1 animals; nevertheless, dystocia was not observed in any treated dams in the P2 generation and one control P2 dam exhibited dystocia.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

One mid-dose male (animal 949) was sent to necropsy for animal welfare concerns associated with a probable nasal fracture. The animal exhibited the following clinical signs: red periocular and perinasal soiling, maloccluded incisors and trauma to its muzzle.

Dam 1030 given 300 ppm delivered 13 live and 3 dead pups on GD 22 (LD 0), but then appeared pale, had labored respiration, an ungroomed appearance, a red vulvar discharge and decreased activity on LD 1 and was sent in a moribund condition to necropsy on TD 99 (LD 1). At necropsy, dam 1030 had cecal edema and hemorrhages, hemolyzed blood and ascites within the abdominal cavity, dark kidneys, a pale liver and lungs, stomach ulcers and the uterus contained two dead fetuses and retained placentas.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Male
P1 males had similar body weights across dose groups prior to initiation of dosing. Body weights were decreased by up to 5% in the high-dose P1 males during the pre- and post-mating periods; however, these differences were not statistically significant. P1 male body weight gains were decreased in the high-dose group by 2.5-10% across all intervals after TD 1-2 with statistically significant effects on body weight gains on TD 1-2 (63% decrease). Given the minimal differences from controls, these body weight/gain effects were not considered to be biologically significant. There were no treatment-related effects on body weight or body weight gain at any intervals at lower dose levels of biphenyl.

Female Pre-mating
P1 females had similar body weights across dose groups prior to initiation of dosing. Body weights of high-dose females during the pre-breeding period were statistically identified as decreased (~5-6%) from TD 15-71. Decreases in body weight gain were seen at 2800 ppm with the initiation of biphenyl treatment. Consistent with these body weight decrements, body weight gains relative to TD 1 were significantly decreased by ~15-26% in high-dose females at all intervals from TD 15-71. There were no treatment-related effects on body weight or body weight gains at doses ≤ 1000 ppm biphenyl.

Female Gestation
Body weights in the 2800 ppm group were decreased throughout gestation by
≤ 5.6% (relative to controls) and were statistically identified as decreased relative to controls on GD 7 and 14. There were no treatment-related gestation body weight effects at doses of ≤ 1000 ppm. Although not statistically identified, the GD 0-7 body weight gain in the 2800 ppm group was decreased 14.2% compared to controls. There were no treatment-related gestation body weight gain effects at doses of ≤ 1000 ppm biphenyl.

Female Lactation
During lactation, female body weights in the high-dose group were statistically identified as decreased by 5.2% on LD 4 and by 6.4% on LD 7. These body weight decreases were associated with a decrease in lactation body weight gains on LD 4-7 in the high-dose dams. Lactation body weights were not significantly affected in 2800 ppm dams after LD 7 and were similar across dose groups on LD 21. During the second week of the lactation period, the high-dose dams had a statistically identified increase in lactation body weight gain on LD 7-14. During LD 14-21, dams at all doses lost weight; however, high-dose dams lost less weight, resulting in an overall 36.6% increase (not statistically significant) in lactation body weight gain over the LD 1-21 period. There were no treatment-related lactation body weight gain effects at doses ≤ 1000 ppm through LD 21.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Male
Feed consumption was statistically identified as decreased in P1 males provided with 2800 ppm on test days 1-2 (20.2%), but this effect was transient. Thereafter, high-dose males ate slightly less feed at all intervals than control animals. This resulted in slightly lower body weights (up to 5% less than controls) and lower body weight gains (2.5-10% decrease across all intervals after TD 1-2) during the pre- and post-mating periods. There were no treatment-related differences in P1 male feed consumption during the pre-mating or post-mating period at doses ≤ 1000 ppm.

Female Pre-mating
When compared to controls, females provided 2800 ppm biphenyl in the diet had a treatment-related decrease in pre-mating feed consumption from TD 1-43 with statistically-identified differences noted on TD 1-2, 4-22, and 36-43. These decreases in feed consumption correlated with the decreases in body weight/body weight gain seen in the high-dose P1 females during the pre-mating period. Feed consumption was also statistically identified as decreased on TD 1-2 in females at 1000 ppm. Feed consumption on TD 8-15 was lower in the 300 ppm females and was statistically identified; however, this result was considered incidental as there was no dose-response relationship and no corresponding effect on body weight or body weight gain in the 300 ppm female this group.

Female Gestation
There were no statistically identified differences in gestation feed consumption with biphenyl exposure. Feed consumption was decreased slightly (7.6%) in the high-dose dams on GD 0-7. This decrease in feed consumption correlated with a 14.2% decrease in body weight gain on GD 0-7 seen in the high-dose P1 females during the gestation period. There were no treatment-related effects on gestation feed consumption at biphenyl doses ≤ 1000 ppm.

Female Lactation
There were no statistically identified differences from control at any dose level through LD 21. Lactation feed consumption in high-dose dams was decreased by 8.4% on LD 1-4 and 9% on LD 4-7, which corresponded with intervals of decreased lactation body weights/gains in this dose group. The relative difference in feed consumption improved for high-dose dams thereafter, which may be related to the lower dietary concentrations of biphenyl (one-half concentration) that was introduced on LD 7 and one-third concentration that was introduced on LD 14. There were no treatment-related effects on lactation feed consumption at biphenyl doses ≤ 1000 ppm.


Test Material Intake – P1 Males and P1 and Satellite Females

Targeted dose levels for dietary biphenyl exposures were 25, 75, and 215 mg/kg/day biphenyl for the low-, mid- and high-dose groups, respectively. Throughout the dosing periods (except during the lactation period), rats ate less feed/kg body weight, resulting in higher doses at the start of the dosing period and lower doses at the end of the dosing period. Overall, dietary exposures were less than nominal levels for the P1 generation through gestation, and at or slightly above nominal in the lactation period.

Male
Test material intake in P1 males was 16.0 mg/kg/day at 300 ppm, 54.4 mg/kg/day at 1000 ppm, and 150 mg/kg/day at 2800 ppm.

Female Pre-mating
During the pre-mating period, test material intake in P1 females was 19.4 mg/kg/day at 300 ppm, 65.2 mg/kg/day at 1000 ppm, and 179 mg/kg/day at 2800 ppm.

Female Gestation
The test material intake for P1 females during gestation was 18.2 mg/kg/day at 300 ppm, 60.0 mg/kg/day at 1000 ppm, and 168 mg/kg/day at 2800 ppm.

Female Lactation
The test material intake for P1 females during lactation was 25.4 mg/kg/day at 300 ppm, 86.5 mg/kg/day at 1000 ppm, and 238 mg/kg/day at 2800 ppm.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Hematological parameters were examined in 10 P1 animals/sex/dose group.
Females given 1000 ppm biphenyl had a statistically significant higher mean hematocrit. This hematologic alteration was interpreted to be normal biological variability unrelated to treatment because of the lack of a dose-response relationship. Differential white blood cell counts and prothrombin times were similar across groups for both males and females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Thyroid Hormone Levels – P1 Adults
There were no treatment-related effects in serum T3, T4 or TSH in P1 males and LD 22 dams at any dose of biphenyl in the diet. The P1 female TSH control values had a higher CV due to an outlier value in dam #998. When this value was removed, the mean TSH value for the control group was 8.3 ng/ml with a standard deviation of 3.2. This is similar to values in the biphenyl-treated groups and again indicated no treatment-related change in TSH values.

Clinical chemistry parameters were examined in four satellite females/dose group. The only clinical chemistry alteration was a statistically significant higher triglyceride level of females given 300 ppm dietary biphenyl. This alteration in triglyceride level was interpreted to be unrelated to treatment due to a lack of a dose-response relationship, and was largely attributed to two individual rats in the 300 ppm group (#1042 and #1044). In addition, a change in triglyceride levels was not seen in the main study P1 females where the sample size was considerably larger (Satellite n=4 vs. P1 females n=10).

Clinical chemistry parameters were examined in 10 P1 animals/sex/dose group. There were no treatment-related changes in any of the clinical chemistry parameters in either males or females at any dose level. Statistically identified clinical chemistry alterations were confined to serum alkaline phosphatase and calcium levels in P1 females. A statistically significant, higher serum alkaline phosphatase (ALP) concentration was noted in P1 females given 2800 ppm biphenyl. This alteration in ALP was interpreted to be spurious and unrelated to treatment because there were no statistically identified or treatment-related differences in ALP concentrations in the 2800 ppm satellite females or the 2800 ppm P2 females compared to the controls. Serum calcium concentrations were marginally lower and statistically identified in P1 females given 300 or 2800 ppm. The differences in serum calcium levels were interpreted to be normal variability unrelated to treatment due to lack of a dose-response relationship. Moreover, there were no statistically-identified serum calcium differences in any of the satellite female groups or the P2 female groups compared to the controls. Lastly, one control P1 dam (#998) had high values for GGT (8.1 u/L) and ALT (73 u/L), which were identified as outliers. When these values were removed, the P1 control female GGT value was 1.5 ± 0.0 u/L and its ALT value was 44.1 ± 5.9 u/L, similar to the values reported in the biphenyl-treated groups.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Urinalysis parameters were examined in four satellite females/dose group to assess kidney function in non-pregnant females exposed to biphenyl. The only treatment-related effect on urinalysis parameters was the presence of smooth ovoid crystals with a central depression noted in the pooled urinary sediment of the satellite females given 2800 ppm. The crystals were of various sizes and shapes ranging from approximately 10 to 40 microns in length. The exact nature/composition of the crystals is unclear, however, they likely represent the test material and/or its metabolites.

Urinalysis parameters were examined in 10 P1 males and 4 P1 females/dose group. There were no statistically significant differences in urine volume or specific gravity in males or females between the controls and any of the treatment groups. There were no treatment-related differences in any of the urinalysis parameters of males or females compared to the controls at any dose level.
With microscopic examination, pooled urine sediment of males given 2800 ppm had 10-14 WBC/high power field as compared to ‘rare’ WBC/high power field in the controls. This difference was interpreted as spurious and unrelated to treatment because it was not repeated in Cohort 1A and Cohort 1B P2 2800 ppm males
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:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Frozen sections of the left and right kidneys from four control and four 2800 ppm females were collected and stained with hematoxylin and examined for the presence of precipitates or crystals within the kidney. The purpose of evaluating frozen sections was to preserve possible treatment-related precipitates or crystals which would otherwise be possibly lost by formalin fixation and routine histological processing methods.
Evaluation of the frozen sections under the light microscope as well as under polarized light, however, did not reveal the presence of any treatment-related precipitates or crystals within any of the kidneys of the 2800 ppm satellite females.

Histopathological Observations – P1 Males and P1 Females

The liver and kidney were identified as target organs in the high-dose males and females. In addition, the urinary bladder was also identified as a target organ for the high-dose males.
Although there were no remarkable liver weight differences compared to the controls, males given 1000 ppm and males and females given 2800 ppm had dose-related, increased incidence of a very slight hypertrophy of the centrilobular/midzonal hepatocytes. The hepatocytes contained fine homogeneous cytoplasm with increased eosinophilia as compared to the controls. The hypertrophic change was minimal and was possibly due to enzyme induction. Based on the absence of other treatment-related histopathological findings such as necrosis, inflammation, fibrosis, vacuolation, proliferation, degeneration, etc., in the affected livers, or any clinical chemistry changes indicative of liver injury, the minimal hepatocyte hypertrophy was interpreted to be an adaptive, non-adverse change in response to the continued ingestion of biphenyl.
Treatment-related histopathological changes in the kidneys were noted in males given 2800 ppm and in females given 1000 or 2800 ppm. Within the 2800 ppm males, only a small proportion of rats were affected. The lesions were mostly localized to the renal papilla, renal pelvic epithelium and the distal collecting ducts. The lesions were characterized by very slight or moderate, focal or multifocal hyperplasia of the renal papillary epithelium, slight focal or multifocal hyperplasia of the renal pelvic epithelium, very slight or slight multifocal epithelial hyperplasia and hypertrophy of the collecting ducts in the papilla and slight multifocal subacute to chronic inflammation of the papilla. The hyperplasia of the renal papillary and pelvic epithelium was characterized by very slight or slight thickening of the epithelial cell layer. In the collecting ducts, hyperplasia and hypertrophy of the lining epithelium was characterized by the presence of larger, deeply basophilic cells with occasional mitotic figures with very slight crowding of the cells. The inflammatory change was generally slight in severity and was characterized by a slight increase in the number of mononuclear cells occasionally admixed with small numbers of neutrophils underneath the papillary or renal pelvic epithelium. Other associated treatment-related findings, although less frequent, included very slight multifocal necrosis of the collecting duct epithelium, slight focal edema of the tip of the papilla, slight focal ulceration of the papilla and slight hemorrhage in the renal pelvis. All these histopathological changes were consistent with possible irritant effects of urinary crystals (crystalluria) or microcalculi within the renal pelvis or papillary collecting ducts, although they were only rarely observed in the high-dose P1 males. Among the 2800 ppm P1 males, one male (#971) had small amounts of an eosinophilic to red granular material variably forming small aggregates or clumps consistent with microcalculi admixed with small amounts of blood within the renal pelvis. The nature of this microcalculi is not clear, however, it likely represents urinary precipitates of the test material and/or its metabolites. This finding was interpreted to be treatment-related because similar findings of the same granular microcalculi was noted in some of the Cohort 1A and Cohort 1B P2 males given 2800 ppm. Furthermore, the necropsy findings of treatment-related calculi in the bladders of two P2 males given 2800 ppm (see Pathology section on Cohort 1A and Cohort 1B P2 animals), treatment-related crystals in the urinary sediment of satellite females (see Urinalysis section on satellite females) are consistent with the aforementioned treatment-related kidney findings in the 2800 ppm P1 males. In addition, biphenyl has been previously shown to induce urinary crystals (Ohnishi et al., 2000a; Ohnishi et al., 2001) or bladder calculi in rats (Shiraiwa et al., 1989; Ohnishi et al., 2000b; Umeda et al., 2002).
Females given 1000 or 2800 ppm had dose related incidence and severity of a very slight or slight tubular degeneration localized to the outer stripe of the outer medulla. This treatment-related change was characterized by tubules with very slightly dilated lumens at multiple foci, lined by epithelial cells that variably contained fine cytoplasmic vacuoles and were very slightly reduced in cell height (attenuated) compared to the controls. However, their brush borders were intact and there were no treatment-related necrotic or inflammatory changes. The lumens of these tubules often contained increased amounts of eosinophilic homogeneous or globular material compared to the controls. The pathogenesis of this treatment-related change in the females is not clear, particularly given its absence in the high-dose males. In the urinary bladder, males given 2800 ppm had treatment-related, very slight or slight, simple diffuse urothelial hyperplasia along with a very slight multifocal subacute to chronic inflammation in the lamina propria underlying the urothelial lining of the bladder. The hyperplasia was characterized by very slight or slight uniform thickening of the urothelium (simple hyperplasia) compared to the thickness of the control bladders. The subacute to chronic inflammation was characterized by the presence of small numbers of mononuclear cells within the lamina propria underlying the urothelium. These changes were consistent with chronic irritant effects on the urothelial lining of the bladder by possible urinary crystals or calculi although none were observed grossly or microscopically within these bladders. However, treatment-related bladder calculi were observed in two 2800 ppm P2 males at the scheduled necropsy. Moreover, biphenyl has been previously shown to induce urinary crystals (Ohnishi et al., 2000a; Ohnishi et al., 2001) or bladder calculi in rats (Shiraiwa et al., 1989; Ohnishi et al., 2000b; Umeda et al., 2002).
There were no treatment-related microscopic bladder effects in P1 females given 2800 ppm biphenyl. All other histopathological observations in the liver, kidney, urinary bladder and all other organs examined were spontaneous changes unassociated with the exposure to biphenyl due to isolated occurrences and/or lack of dose-response relationship.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

There was no significant difference in mean estrous cycle length in the biphenyl-treated groups compared with the control group. The mean estrous cycle length for all groups ranged from 4.0-4.2 days. There was no indication of persistent estrus (i.e., greater than two consecutive days in estrus) in either the P1 control or biphenyl-treated animals. There was no apparent difference in the percentage of time spent in estrus in biphenyl-treated females compared with controls. Although, there was an apparent increase in the percentage of time spent in diestrus in some 2800 ppm biphenyl treated females compared with controls, this difference was due to dams 1076, 1091, 1093 and 1098 that were pregnant (littered) without apparent evidence of mating and therefore appeared to be in prolonged diestrus.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Sperm Motility

There were no significant, treatment-related effects on sperm motility or progressive motility when P1 males were exposed to concentrations of < 2800 ppm biphenyl in the diet. One control male (#895) had no motile sperm. During pathological examination, this male was found to have an extensive sperm granuloma in the cauda epididymis. When this animal was removed from the group, the control P1 male values were 84.7 ± 7.2% for motile sperm and 69.2 ± 8.1 % for progressively motile sperm. These values were similar to sperm motility values in the biphenyl-treated P1 males.

Sperm Counts

Examination of epididymal sperm counts of the P1 control and high-dose males initially revealed a statistically significant decrease in the high-dose male sperm concentration per gram of epididymis but not total sperm count, thus triggering an analysis of the 300 and 1000 ppm dose groups. Subsequent analysis of the combined data from all groups indicated that the decrease at the high-dose was no longer statistically significant. In addition, there were no treatment-related effects on epididymal total sperm count at any tested dose, nor were any effects observed in the second generation Cohort 1B (P2) males or Cohort 1A males. Also, there were no corroborating effects on testicular or epididymal weights or histopathology. Analysis of individual animal data in the high dose group revealed two males (#969 and 977) with low sperm conc/g tissue values of 498.9 and 598.7 (x10E6) that played a role in decreasing the mean values. Nevertheless, these males had sperm motility, progressive motility, and percentage of abnormal sperm comparable to controls and sired normal litters; therefore, there was no compromise in fertility. When comparing the results to laboratory historical control, P1 epididymal total sperm count and sperm concentration values were within the historical control range. Taken together, this weight of evidence indicated a lack of a treatment-related effect on sperm counts.
Reproductive performance:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Reproductive Indices, Pup Survival, and Sex Ratio – P1/F1 Animals

Reproductive Indices

Dietary exposure to biphenyl had no treatment-related effect on any of the reproductive indices, including male and female mating, conception, fertility, gestation indices, or percent post-implantation loss.

Time to Mating and Gestation Length

Dietary exposure to biphenyl had no treatment-related effect on time to mating or gestation length.

Sex Ratio

Dietary exposure to biphenyl had no treatment-related effect on offspring sex ratio.

Offspring Survival

Although the gestation survival index was slightly lower and statistically identified in the 2800 ppm group (98.0%) when compared to control (100%), this slight difference was deemed unrelated to treatment because it was within the laboratory historical control range and did not exhibit a dose-response relationship.
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
2 800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Cohort 1B P2 males and females did not exhibit any treatment-related observations throughout the term of the study. Clinical observations recorded during the study were isolated, spontaneous occurrences common to this strain/age of rat and unrelated to exposure. One control female (#2496) was found with an injured/inflamed lower hindlimb and was sent to necropsy for animal welfare concerns. One control female (animal #2503) died spontaneously on TD 146 (LD 7) due to acute mastitis induced sepsis. This animal exhibited the following clinical signs on LD 6 prior to death: periocular soiling, shallow/rapid respiration, a mammary enlargement, a lame gait, decreased activity, swelling of its neck and muzzle and partially closed eye lids. Also, one control female (animal #2508) was euthanized in moribund condition due to dystocia on TD 141 (GD 23). The dam delivered three dead pups in the cage and exhibited the following clinical signs: difficult birth, pale skin/mucous membranes, labored respiration, cold to touch, eye lids partially closed, and decreased activity. None of these deaths were attributed to biphenyl treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

One control female (#2496) was found with an injured/inflamed lower hindlimb and was sent to necropsy for animal welfare concerns. One control female (animal #2503) died spontaneously on TD 146 (LD 7) due to acute mastitis induced sepsis. This animal exhibited the following clinical signs on LD 6 prior to death: periocular soiling, shallow/rapid respiration, a mammary enlargement, a lame gait, decreased activity, swelling of its neck and muzzle and partially closed eye lids. Also, one control female (animal #2508) was euthanized in moribund condition due to dystocia on TD 141 (GD 23). The dam delivered three dead pups in the cage and exhibited the following clinical signs: difficult birth, pale skin/mucous membranes, labored respiration, cold to touch, eye lids partially closed, and decreased activity. None of these deaths were attributed to biphenyl treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Male
Cohort 1B P2 high-dose males had significantly decreased (5.7-8.8%) body weights from PND 56 through PND 135. Body weight gains were significantly decreased (6.2-9.4%) in high-dose animals at all intervals from PND 56 through PND 135. The effects on body weights/body weight gains in the 2800 ppm Cohort 1B P2 males were similar to the effects seen in the Cohort 1A males at this same dose level. Thus, the effects on body weight/body weight gain in the 2800 ppm Cohort 1B P2 males were interpreted to be treatment related. There were no treatment-related effects on body weight or body weight gains at doses ≤ 1000 ppm biphenyl.

Female Pre-mating
Similar to the Cohort 1A females, there were no stastistically significant decreases in body weights of high-dose Cohort 1B P2 females at any time points during the premating exposure period. High-dose Cohort 1B P2 females weighed 5.3% less than controls on PND 28, which was sustained throughout the premating period (ranging 5.1-6.8% from PND 70-105). There were no treatment-related effects on body weights at biphenyl doses ≤ 1000 ppm. Although not statistically identified, body weight gains of high dose Cohort 1B P2 females were were slightly decreased (5.6-7.5%) relative to controls from PND 70-105. There were no treatment-related effects on body weight gain at biphenyl doses ≤ 1000 ppm.

Female Gestation
Although not statistically identified, body weights in the 2800 ppm group P2 females were decreased throughout gestation by (≤ 6.7% relative to controls). There were no treatment-related gestation body weight effects at biphenyl doses of ≤ 1000 ppm. Although not statistically identified, the GD 0-7 body weight gain in the 2800 ppm group was decreased 17% compared to controls. The statistically identified increase in gestation body weight gain in the 300 ppm dose group was considered spurious and unrelated to treatment. There were no treatment-related gestation body weight gain effects at biphenyl doses of ≤ 1000 ppm.

Female Lactation
During lactation, P2 female body weights in the high-dose group were significantly decreased by 8.0% on LD 4 and by 8.3% on LD 7. These body weight decreases were associated with decreases in lactation body weight gains on LD 1-4 and 4-7 in the high-dose dams. Lactation body weights were not significantly affected in 2800 ppm dams after LD 7 and were similar across dose groups on LD 21. The statistically identified increase in lactation body weights on LD 4, 7 and 21 in the 300 ppm dose group was considered spurious and unrelated to treatment. During the second week of the lactation period, the high-dose dams had a statistically identified increase in lactation body weight gain on LD 7-14. During LD 14-21, dams at all doses lost weight; however, high-dose dams lost less weight, resulting in a statistically identified 114.0% increase in lactation body weight gain (LD 1-21) in the high-dose dams. This period corresponded to the administration of half (LD 7-14) followed by one third (LD 14-21) the dietary concentration of test material and a concomitant improvement in feed consumption for the high dose group. There were no treatment-related lactation body weight body weight gain effects at doses ≤ 1000 ppm through LD 21.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

In high-dose Cohort 1B P2 males, feed consumption was decreased significantly from PND 35-98, which coincides with the periods of decreased body weights and body weight gains in these animals. Feed consumption was not recorded after day 105 due to co-housing of P2 males and females. Feed consumption was not significantly altered in Cohort 1B P2 males given ≤ 1000 ppm biphenyl.

Female Pre-mating
There were significant treatment-related decreases in Cohort 1B P2 female feed consumption during the post-weaning intervals on PND 70-91. These decreases in feed consumption were consistent with the decreases in body weight/body weight gain seen in the high-dose Cohort 1B P2 females. Feed consumption was not significantly altered in Cohort 1B P2 females given < 1000 ppm biphenyl at any time interval.

Female Gestation
There were no statistically identified differences in gestation feed consumption with biphenyl exposure. Feed consumption was decreased slightly (6.5%) in the high-dose dams on GD 0-7. There were no treatment-related effects on gestation feed consumption at biphenyl doses ≤ 1000 ppm.

Female Lactation
There were no statistically identified differences from control at any dose level through LD 21. Lactation feed consumption in high-dose dams was decreased by 8.6% on LD 4-7, which corresponded with an interval of decreased lactation body weights/gains in this dose group. The relative difference in feed consumption improved for high-dose dams thereafter, which may be related to the lower dietary concentrations of biphenyl (one-half concentration) that was introduced on LD 7 and one-third concentration that was introduced on LD 14. There were no treatment-related effects on lactation feed consumption at biphenyl doses ≤ 1000 ppm.

Test material intake (time weighted average) in Cohort 1B P2 males was 21.6 mg/kg/day at 300 ppm, 72.3 mg/kg/day at 1000 ppm and 204 mg/kg/day at 2800 ppm. As with the Cohort 1A males, the highest intakes of test material in males were recorded for PND 35-42, just after they received their full dietary concentrations and were 35.0 mg/kg/day at 300 ppm, 118 mg/kg/day at 1000 ppm, and 329 mg/kg/day at 2800 ppm.

Female Pre-mating
During the pre-mating period, test material intake (time weighted average) in Cohort 1B P2 females was 23.1 mg/kg/day at 300 ppm, 78.7 mg/kg/day at 1000 ppm, and 218 mg/kg/day at 2800 ppm. As with the Cohort 1A females, the highest intakes of test material in females were recorded for PND 35-42, just after they received their full dietary concentrations and were 33.7 mg/kg/day at 300 ppm, 113 mg/kg/day at 1000 ppm, and 322 mg/kg/day at 2800 ppm.

Female Gestation
The test material intake for Cohort 1B P2 females during gestation was 19.3 mg/kg/day at 300 ppm, 64.9 mg/kg/day at 1000 ppm, and 176 mg/kg/day at 2800 ppm.

Female Lactation
The test material intake for Cohort 1B P2 females during lactation was 25.0 mg/kg/day at 300 ppm, 88.5 mg/kg/day at 1000 ppm, and 255 mg/kg/day at 2800 ppm. With dietary concentration adjustments prior to PND 35, Cohort 1B P2 animals received doses of biphenyl (mg/kg body weight) during lactation that ranged from approximately 28.9-42.5% higher than the doses received by P1 pre-mating female adults. The Cohort 1B P2 male and female (premating) test material intake was closer to the Cohort 1A animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Hematological parameters were examined in 10 Cohort 1B P2 animals/sex/dose level. Males given 300 ppm had a higher reticulocyte count as compared to controls. The higher reticulocyte count was interpreted to be unrelated to treatment, due to lack of any red blood cell changes, absence of a clear dose response in males, and no effect in the Cohort 1A males or Cohort 1B P2 females. All other hematological parameters, including differential WBC counts and prothrombin times, were similar to controls across all dose groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Clinical chemistry parameters were examined in 10 Cohort1 B P2 animals/sex/dose group. P2 males given 2800 ppm had statistically identified, marginally (6%) lower serum potassium concentration compared to the controls that was interpreted to be unrelated to treatment due to the minimal difference from the control, absence of such findings in P1 or Cohort 1A males or females or P2 females.

Serum urea nitrogen was statistically identified as higher in females given 1000 ppm, however, it was interpreted to be spurious and unrelated to treatment due to lack of a dose-response relationship. Serum phosphorous concentration was slightly higher (14.7%) in P2 females given 2800 ppm and was interpreted to be treatment-related, although the exact mechanism is not clear.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Urinalysis parameters were examined in 10 Cohort 1B P2 males/sex/dose group and 4 Cohort 1B P2 females/sex/dose group. Males given 2800 ppm had higher urine volumes and statistically identified lower specific gravity. The higher urine volume and lower specific gravity in males given 2800 ppm were interpreted to be treatment-related. All male groups including control had evidence of blood in the urine (hematuria) in some of the rats. It is not uncommon to have trace quantities of blood in occasional control rats likely due to spontaneous injury, inflammation, or calculi in the urinary tract. However, the severity of hematuria was slightly higher in males given 2800 ppm compared to the controls (4 of 10 high-dose males had 3+ severity versus 3 of 10 control males had 1+ severity) which was interpreted to be treatment-related. Urinary microsediment of males given 2800 ppm had amorphous phosphate crystals which were not present in the controls and the middle- and low-dose groups, and thus, its presence was considered related to treatment.

In P2 females, there were no statistically identified, volume or specific gravity changes in the 2800 ppm group females compared to the controls. While control females were mostly negative for hematuria (3 of 4 negative and 1 of 4 with 1+ severity) all high-dose females (4 of 4) had evidence of hematuria (trace quantities in 3 of 4, and a 2+ severity in 1 of 4) and hence, was considered treatment-related. The source of the blood, whether it was from the kidney, urinary bladder or the genital tract could not be definitively ascertained. Unlike the P2 males, no crystals of any kind were noted in the urinary sediment of females of any dose group including the control.
All other urinary parameters of Cohort 1B P2 males and females exposed to biphenyl were interpreted to be spontaneous alterations due to isolated occurrences and/or lack of a dose-response relationship.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Male
P2 males given 2800 ppm had a 7.9% decrease in their final body weights, which was interpreted to be treatment related. Males given 1000 or 2800 ppm had treatment-related higher relative liver weights compared to control; however, the differences were statistically significant only in the 2800 ppm males. Absolute liver weights were also slightly higher in the 1000 or 2800 ppm males; however, their differences from control did not reach statistical significance. The higher liver weights of the 1000 or 2800 ppm males were interpreted to be treatment-related as they corresponded to microscopic finding of treatment-related hepatocyte hypertrophy. There were no treatment-related effects on the absolute or relative weights of the kidneys, prostate, testes, seminal vesicles with coagulating glands, epididymides or pituitary at any dose of biphenyl.

Female
The mean final body weight of females given 2800 ppm was 2.9% lower than controls, but was not statistically identified.
There was a slight treatment-related increase in absolute and relative kidney weights at the high dose. The higher mean relative kidney weight of the high-dose females was statistically identified compared to the controls. In addition, absolute and relative liver weights were slightly increased compared to controls, which corresponded to treatment-related hepatocyte hypertrophy. The higher mean relative liver weight of the high-dose females were statistically identified compared to the control. These higher organ weights were likely manifest only in the P2 females due to the longer duration of exposure in comparison to the P1 and Cohort 1A females.
There were no treatment-related effects on the absolute or relative weights of the pituitary gland, adrenal gland, thyroid gland, ovaries, or uterus at any dose of biphenyl.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

The only organ with treatment-related gross pathological observations was the urinary bladder. Two males (#2470 and #2478) given 2800 ppm had tan-grey, ovoid, firm, calculi within the bladder. Each calculus was approximately 6x4 mm or 7x5 mm in size. In addition, the bladder walls were thickened in these two rats. These gross findings of urinary bladder calculi and thickening of the bladder wall were interpreted to be treatment-related. All other gross pathological observations were interpreted to be spontaneous alterations, unassociated with the dietary administration of biphenyl.

Similar to the P1 females, focal or multifocal erosions/ulcers were noted in the glandular mucosa of the P2 females across all dose groups including the control. However, their incidences were not dose-related and therefore, these were interpreted to be spontaneous changes unrelated to biphenyl exposure and were attributed to non-specific stress (Nolte et al., 2016) likely associated with lactation and overnight fasting prior to necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

The liver, kidney, and the urinary bladder were identified as target organs for males and females in the Cohort 1B P2 group.
Treatment-related changes in the liver of males and females given 1000 or 2800 ppm consisted of a very slight or slight hypertrophy of the centrilobular/midzonal hepatocytes with increased cytoplasmic eosinophilia. The hepatocyte hypertrophy corresponded with increased relative liver weights for the 2800 ppm males and females. Unlike the P1 and Cohort 1A groups, the severity of hepatocyte hypertrophy was higher in 2800 ppm Cohort 1B males (‘slight’ versus ‘very slight’ in P1 and Cohort 1A 2800 ppm males) as well as the occurrence of very slight hepatocyte hypertrophy in 1000 ppm female group that is consistent with the increased duration of biphenyl exposure. The affected hepatocytes contained fine homogeneous cytoplasm with increased cytoplasmic eosinophilia compared to the controls. There were no other treatment-related changes in the liver such as necrosis, inflammation, fibrosis, vacuolation, proliferation, degeneration etc., in the affected livers, nor were there any indications of liver injury from the clinical chemistry data. Therefore, the very slight or slight hepatocyte hypertrophy was interpreted to be an adaptive non-adverse change associated with the continued ingestion of biphenyl.

Treatment-related histopathological changes in the kidneys were noted in males given 2800 ppm and in females given 1000 or 2800 ppm. As noted with the P1 and Cohort 1A high-dose males, only a few males given 2800 ppm had treatment-related changes. In 5 of 21 males given 2800 ppm, there were small to moderate amounts of an eosinophilic or red granular or clumped urinary precipitates/microcalculi admixed with red blood cells in the renal pelvis representing treatment-related very slight or slight hemorrhage in the pelvis in 4 high-dose males. A slight multifocal treatment-related hemorrhage in the medullary tubules and collecting ducts was also noted in one male given 2800 ppm likely due to the presence of these precipitates/microcalculi within these tubules. The exact nature of these urinary precipitate/microcalculi was not clear, however, it likely represented the test material and/or its metabolites. Consistent with the presence of these precipitates/microcalculi, the treatment-related lesions were mostly localized to the renal papilla, renal pelvic epithelium and the distal collecting ducts. The treatment-related changes were characterized by slight or moderate focal or multifocal hyperplasia of the renal papillary epithelium, very slight focal or multifocal hyperplasia of the renal pelvic epithelium, very slight multifocal epithelial hyperplasia and hypertrophy of the collecting ducts in the papilla, and slight focal or multifocal subacute to chronic inflammation of the papilla and very slight focal or multifocal subacute to chronic inflammation of the surrounding pelvic epithelium. The hyperplasia of the renal papillary and pelvic epithelium were characterized by very slight or slight thickening of the epithelial cell layer. In the collecting ducts, hyperplasia and hypertrophy of the lining epithelium was characterized by the presence of larger, deeply basophilic cells with occasional mitotic figures with very slight crowding of the cells. The inflammatory change was generally very slight or slight in severity and was characterized by the presence of very slight or slight increase in mononuclear cells underneath the papillary or renal pelvic epithelium.

All other microscopic observations in male kidneys were interpreted to be spontaneous background changes unassociated with the exposure to biphenyl.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

There was no significant difference in mean estrous cycle length in the biphenyl-treated groups compared with the control group. The mean estrous cycle length for all groups was 4.1 days. There was no indication of persistent estrus (i.e., greater than two consecutive days in estrus) in either the P2 control or biphenyl-treated animals. There was no apparent difference in the percentage of time spent in estrus in biphenyl-treated females compared with controls.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Sperm Parameters – Cohort 1B P2 Males

Sperm Motility
There were no significant, treatment-related effects on sperm motility or progressive motility when Cohort 1B P2 males were exposed to concentrations of < 2800 ppm biphenyl in the diet through critical windows of development from in utero through adulthood.

Sperm Counts

There were no significant, exposure-related differences in epididymal sperm and testicular spermatid counts between 0 and 2800 ppm males. Sperm/spermatid counts were not conducted for the lower-dose levels due to the lack of effect at the highest dose.

Sperm Morphology

The proportion of abnormal sperm was not significantly different between control and biphenyl-treated Cohort 1B P2 males. Values less than 5.0% are considered typical for control male rats (Stump et al., 2012; Linder et al., 1992).
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Dietary exposure to biphenyl had no significant effect on any of the reproductive indices, including male and female mating, conception, fertility, and gestation indices, or percent post-implantation loss. Although the 2800 ppm male and female mating, conception and fertility indices were lower than controls, these differences were deemed unrelated to treatment because 1) none of the results were statistically identified; and 2) the P2 male and female mating, conception and fertility indices were near or within the range of the P1 adults including the P1 control animals.

Dietary exposure to biphenyl had no treatment-related effect on time to mating or gestation length
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
2 800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Clinical signs:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Cohort 1A Reproductive Toxicity and Systemic Toxicity Group

In-Life Observations – Cohort 1A Males and Females

Cohort 1A males and females did not exhibit any treatment-related observations throughout the term of the study. Clinical observations recorded during the study were isolated occurrences and/or deemed to be spontaneous occurrences common to this strain/age of rat and unrelated to exposure. One mid-dose female had a persistent vaginal thread recorded during the monitoring period. This observation was deemed unrelated to treatment since it was an isolated occurrence and was not observed in the high-dose females.

Detailed Clinical Observations – Cohort 1A Males and Females

Cohort 1A males and females did not exhibit any treatment-related observations throughout the term of the study.
Dermal irritation (if dermal study):
not examined
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Male
There were no significant decreases in body weights of high-dose Cohort 1A males at early time points during the post-weaning period from PND 21-35; however, from PND 36-84, body weights in the high-dose males were significantly decreased (7.5-9.7%) relative to the controls at all time points through the remainder of the study. Consistent with these body weight decrements, body weight gains were significantly decreased by 8.8-11.1% in high-dose males at all intervals from PND 35-84. The decreases in body weights/body weight gains starting on PND 35 are consistent with the return to full dietary concentrations of biphenyl (from half the normal concentration). Thus, 2800 ppm biphenyl resulted in sustained, treatment-related effects on body weights/body weight gains in Cohort 1A males. There were no treatment-related effects on either body weight or body weight gains at ≤ 1000 ppm biphenyl.

Female
There were no significant decreases in body weights of high-dose Cohort 1A females at any time points during the exposure period. High-dose Cohort 1A females weighed approximately the same as controls on PND 21 and remained similar to controls through PND 28, but there was a slight decrease in body weight by PND 35. Body weights were decreased approximately 5% from PND 36-42 and improved thereafter such that by PND 84, body weights in high-dose females were similar to controls. PND 35 coincided with the re-introduction of full dietary concentrations of biphenyl (from half normal concentration); thus PND 35-42 appears to be an adjustment period for Cohort 1A females. There were no treatment-related body weight effects at biphenyl doses ≤ 1000 ppm. Body weight gains were statistically identified as decreased (7.1 and 7.5%, respectively) from PND 21 to PND 35 and 36 in the high-dose Cohort 1A females and improved thereafter. There were no treatment-related effects on body weight gain at biphenyl doses ≤ 1000 ppm.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Male
In high-dose males, feed consumption values were statistically identified as decreased (7.6-16.9%) from PND 28-84. These decreases in feed consumption were consistent with the decreases in body weight/body weight gain seen in the high-dose Cohort 1A males from PND 35 to PND 84. The PND 35-42 period is just after the animals received their full dietary concentrations and this period corresponded to the period of highest test material intake during which intake for high dose males ranged 306-338 mg/kg/day at 2800 ppm. There were no treatment-related effects on feed consumption in Cohort 1A males given < 1000 ppm biphenyl.

Female
There were significant treatment-related decreases in Cohort 1A female feed consumption during the early post-weaning intervals on PND 28-38. These decreases in feed consumption were consistent with the decreases in body weight/body weight gain seen in the high-dose Cohort 1A females from PND 35 to PND 42 and corresponded to the start of administration of full concentration of biphenyl after the peripubertal period. There were no treatment-related effects on feed consumption in Cohort 1A females given < 1000 ppm biphenyl at any time interval.

Test material intake (time weighted average) in Cohort 1A males was 23.8 mg/kg/day at 300 ppm, 80.3 mg/kg/day at 1000 ppm, and 221 mg/kg/day at 2800 ppm. The highest intakes of test material in males were recorded for PND 35-42, just after they were switched from receiving one third the concentration to receiving the full dietary concentrations and ranged from 33.3-36.3 mg/kg/day at 300 ppm, 113-123 mg/kg/day at 1000 ppm, and 306-338 mg/kg/day at 2800 ppm. Test material intake (time weighted average) in Cohort 1A females was 25.2 mg/kg/day at 300 ppm, 85.6 mg/kg/day at 1000 ppm, and 232 mg/kg/day at 2800 ppm. The highest intakes of test material in females were recorded for PND 35-42, ranging from 32.9-37.0 mg/kg/day at 300 ppm, 111-124 mg/kg/day at 1000 ppm, and 304-332 mg/kg/day at 2800 ppm.
Despite dietary concentration adjustments prior to PND 35, Cohort 1A animals received 29.6-48.8% higher doses of biphenyl (mg/kg body weight) over the entire dosing period than P1 pre-mating (male and female) adults.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Hematological parameters were examined in 10 Cohort 1A (PND 90) animals/sex/dose level. There were no treatment-related changes in any of the hematology parameters in the Cohort 1A males or females at any dose level.
Males given 300 ppm had slightly lower, statistically identified mean RBC count and hematocrit, and higher platelet count as compared to controls. These minor differences were interpreted to be unrelated to treatment, due to the lack of a clear dose response in males, and no effect in the Cohort 1A females or in Cohort 1B males or females, which were dosed for a longer period. Cohort 1A females given 1000 and 2800 ppm biphenyl had statistically identified lower WBC counts, which were interpreted to be unrelated to treatment due to lack of a dose-response relationship, and no effect in the Cohort 1A males or Cohort 1B males or females that were exposed for a longer period. All other hematological parameters, including differential WBC counts and prothrombin times, were similar to controls across all dose groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Clinical chemistry parameters were examined in 10 Cohort 1A animals/sex/dose group. There were no statistically identified or treatment-related effects on any clinical chemistry parameters in Cohort 1A males or females at any dose of biphenyl.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Urinalysis parameters were examined in 10 Cohort 1A animals/sex/dose group.

The only treatment-related change noted in the urinalysis parameters of 2800 ppm males was the presence of amorphous phosphate crystals in the pooled urinary microsediment which were not observed either in the controls or the low and mid-dose groups. Amorphous phosphates are ill-defined aggregates of crystals, while triple phosphate crystals are prism shaped and commonly found in rat urinary microsediments. It is not clear whether the amorphous phosphate crystals represent part of the urinary metabolites of biphenyl and/or related to the treatment-related microcalculi noted within the renal pelvis and/or bladder calculi in some of the 2800 ppm male rats. As this observation was repeated in the P2 males given 2800 ppm the presence of these amorphous phosphate crystals in urinary microsediment of males given 2800 ppm was interpreted to be treatment-related. Moreover, biphenyl has been previously shown to induce urinary crystals. Other urine observations of 2800 ppm males such as, cloudy urine in 10 of 10 males were considered spurious and unrelated to treatment because they were not repeated in the P2 generation.
Sexual maturation:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Puberty Onset – F1 Male and Females (Cohorts 1A, 1B, and 2A, and 3)


Male

There was no evidence of retained preputial threads and no treatment-related effects on age/body weight at preputial separation for biphenyl doses ≤ 2800 ppm. There was a statistically identified decrease in age at preputial separation at 300 ppm biphenyl; however, this result is believed to be incidental. There is no dose-response relationship and the slightly lighter body weight in the 300 ppm males is consistent with their age (i.e., not enhanced growth). Lastly, it is difficult to accelerate preputial separation in male rats, except with potent androgens or 5-alpha-reductase inhibitors (Marty et al., 2001). Biphenyl does not fit the effect profiles for either of these classes of compounds.

Female

There were no treatment-related effects on age/body weight at vaginal opening for biphenyl doses ≤ 2800 ppm. A single incidence of a vaginal thread was noted in one mid-dose female (2367).

Interval Between Vaginal Opening and First Estrus – Cohort 1A Female Offspring

There were no significant differences in the interval between vaginal opening and first estrus in any biphenyl-treated group (2.0-2.7 days) relative to the control group (1.8 days). In many animals, first estrus coincided with vaginal opening.

Mean Estrous Cycle Length and Pattern – Cohort 1A Female Offspring

There was no significant difference in mean estrous cycle length in the biphenyl-treated groups compared with the control group. The mean estrous cycle length ranged from 4.0-4.2 days across all groups. There was no indication of persistent estrus (i.e., greater than 2 consecutive days in estrus) in either the control or biphenyl-treated animals.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Males

Males given 2800 ppm had a treatment-related, statistically significant 10.0% lower final body weight relative to controls.

The weights of the following organs were statistically different from controls, but these organ weight changes were deemed unrelated to treatment and/or not considered toxicologically significant.

Absolute brain weight was decreased slightly (4%) in high-dose males, but relative brain weight was increased (6.6%, not statistically significant); therefore, this finding was attributed to the lower terminal body weights in the high-dose animals. Brain weight has previously been shown to be conserved in the presence of body weight changes (OECD, 2001), and as expected, relative brain weights were increased in response to body weight changes in this study. Brain weights were decreased in P1 males, but were not in the high-dose Cohort 1B P2 males at any dose level (n = 21-23/dose). Note that the Cohort 1B P2 males had a longer exposure period, but also had longer to recover from body weight decrements than the P1 males sacrificed on PND 90. Furthermore, there were no effects on brain weight in the high-dose Cohort 1A females and no associated histopathological changes in either high-dose P1 or Cohort 1A males or females.

The absolute epididymal weights were significantly decreased in Cohort 1A males in the 2800 ppm group. Relative epididymal weights were similar to the control group in all biphenyl-treated Cohort 1A males. Terminal body weights in Cohort 1A males given 2800 ppm biphenyl were decreased by 10%. Previous studies have shown that epididymal weights are often spared in the presence of moderate body weight changes (Carney et al., 2004; Chapin and Gulati, 1997); however, a feed restriction study by Rehm et al. (2008) has shown decreases in absolute epididymal weights with sustained effects on body weight. There were no effects on epididymal weights in the high-dose P1 or Cohort 1B P2 males, which were exposed for a longer period, but evaluated at a later timepoint (i.e., longer to recover from body weight decrements). There were no effects on other reproductive parameters in the P1 or Cohort 1A males, including no treatment-related effects on male reproductive indices, sperm motility, sperm morphology and sperm counts (all collected from the epididymis), and no corresponding histopathological changes in either the testes or epididymides in P1 or Cohort 1A males. Thus, changes in absolute epididymal weights were judged to be not toxicologically significant.

The mean relative kidney weight of males given 2800 ppm was slightly higher, compared to the controls. This difference was interpreted to be secondary to the lower final body weight of the 2800 ppm males.

Males given 2800 ppm had statistically identified lower prostate weights with no change in their relative weights compared to control. Per the guideline recommendations, the prostate glands were further sectioned into dorsolateral lobe and the ventral lobes and weighed separately to detect any potential weight differences attributed to a particular lobe. There were no statistically identified weight differences in either of these prostate lobes compared to their corresponding control, indicating no specific endocrine alterations. Moreover, there were no treatment-related histopathological changes in either of the prostate lobes and hence, the minor weight difference in the whole prostate gland of the 2800 ppm males was interpreted to be secondary to their lower final body weights.
Absolute testes weights of males given 2800 ppm were slightly lower (5.9%) compared to controls with no change in the relative testes weights. These changes in absolute testes weights were considered spurious because there was no pattern of anti-androgenic effects in biphenyl-exposed animals and the decrease in absolute testes weights was not present in the P1 males nor the Cohort 1B P2 males (F1 offspring) despite a similar decrease in final body weights in the P2 males (8.9% compared to controls). Moreover, there were no treatment-related histopathological changes in the testes of Cohort 1A, P1 or Cohort 1B P2 males in the 2800 ppm group nor were any treatment-related changes in the sperm parameters.
There were no treatment-related effects on the absolute or relative weights of the adrenal glands, heart, liver, pituitary, spleen, thymus, thyroid gland, dorsolateral prostate, ventral prostate, seminal vesicles at any dose of biphenyl. In addition, the weights of mesenteric lymph nodes (draining nodes for dietary route of administration) and those of the submandibular lymph nodes (distant nodes) did not reveal any statistically identified or treatment-related differences.

Females

The mean final body weight of females given 2800 ppm was 2.9% lower than controls, but was not statistically identified.
There were no treatment-related effects on the absolute or relative weights of the adrenal glands, heart, kidneys, liver, brain, pituitary, spleen, thymus, thyroid gland, ovaries, or uterus at any dose of biphenyl. In addition, the weights of mesenteric lymph nodes (draining nodes for dietary route of administration) and those of the submandibular lymph nodes (distant nodes) did not reveal any statistically identified or treatment-related differences.
Although there was a statistically identified increase (18.7%) in absolute weight of the ovaries in Cohort 1A females given 300 ppm biphenyl, this difference was not considered treatment-related due to a lack of a dose response and the absence of supporting data (i.e., no effect on puberty onset, estrous cycle, mating/fertility of the Cohort 1B, etc.).
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Histopathological Observations – Cohort 1A Males and Females – PND 90

As noted with the P1 adults, the liver and kidney were identified as target organs for the Cohort 1A males and females. In addition, the urinary bladder was also a target organ for the high-dose males.

Males given 1000 ppm or 2800 ppm and females given 2800 ppm had dose-related increased incidences of treatment-related, very slight hypertrophy of the centrilobular/midzonal hepatocytes with increased cytoplasmic eosinophilia. However, there were no remarkable increases in liver weights as compared to the controls. The affected hepatocytes contained fine homogeneous cytoplasm with increased eosinophilia as compared to the controls. The hypertrophic change was minimal and there were no other treatment-related histopathological findings such as necrosis, inflammation, fibrosis, vacuolation, proliferation, degeneration, etc., in the affected livers. In addition there were no clinical chemistry changes indicative of liver injury, Therefore, the very slight hepatocyte hypertrophy was interpreted to be a non-adverse adaptive change in response to the continued ingestion of biphenyl.

Treatment-related histopathological changes in the kidneys were noted in males given 2800 ppm and in females given 1000 or 2800 ppm. As noted with the P1 high-dose males, only a small proportion of males given 2800 ppm were affected. Small amounts of an eosinophilic or red granular to aggregated urinary microcalculi were noted in the renal pelvis of two rats given 2800 ppm along with very slight hemorrhage in the pelvis in one of these rats. The exact nature of these urinary microcalculi are not clear, however, it likely represents the test material and/or its metabolites. Consistent with the presence of these microcalculi, the treatment-related lesions were mostly localized to the renal papilla, renal pelvic epithelium and the distal collecting ducts. The lesions were characterized by very slight or slight, focal or multifocal hyperplasia of the renal papillary epithelium, very slight or slight, focal or multifocal hyperplasia of the renal pelvic epithelium, very slight or slight multifocal epithelial hyperplasia and hypertrophy of the collecting ducts in the papilla, and slight multifocal subacute to chronic inflammation of the papilla and pelvic epithelium. The hyperplasia of the renal papillary and pelvic epithelium were characterized by very slight or slight thickening of the epithelial cell layer. In the collecting ducts, hyperplasia and hypertrophy of the lining epithelium was characterized by the presence of larger, deeply basophilic cells with occasional mitotic figures with very slight crowding of the cells. The inflammatory change was generally very slight or slight in severity and was characterized by the presence of very slight or slight increase in mononuclear cells underneath the papillary or renal pelvic epithelium.

Similar to the P1 females, Cohort 1A females given 1000 or 2800 ppm had a dose-related increased incidence of very slight tubular degeneration localized to the outer stripe of the outer medulla. This treatment-related change was characterized by tubules with very slightly dilated lumens lined by epithelial cells that variably contained fine cytoplasmic vacuoles and were very slightly reduced in cell height (attenuated) compared to the controls. However, their brush borders were intact and there was no treatment-related necrotic or inflammatory changes. The lumens of these tubules often contained increased amounts of eosinophilic homogeneous or globular material compared to the controls. In addition to the very slight tubular degeneration described above, there was treatment-related increased incidence and severity of multifocal medullary tubular mineralization in females given 1000 or 2800 ppm compared to the controls. While the medullary tubular mineralization of affected controls were graded as ‘very slight’, it was graded as ‘slight’ in most of the affected 1000 ppm and 2800 ppm females, based on the extent of distribution and size of the mineralized foci. The mineralization was often present in the outer stripe or at the junction between the outer stripe and the inner stripes of the renal outer medulla. Rarely, in some females given 2800 ppm, there was very slight or slight, focal or multifocal granulomatous inflammation characterized by the presence of small numbers of mononuclear cells, macrophages and multinucleated giant cells surrounding the mineralized foci. The treatment related medullary tubular mineralization noted in the Cohort 1A females given 1000 or 2800 ppm was interpreted to be due to longer duration of exposure and/or higher dose of biphenyl in comparison to the P1 females.
In the urinary bladder, males given 2800 ppm had treatment-related, very slight or slight simple diffuse urothelial hyperplasia along with a very slight or slight multifocal subacute to chronic inflammation. The inflammation was characterized by the presence of slightly increased numbers of mononuclear cells in the lamina propria underlying the urothelial lining of the bladder. The hyperplasia very slight or slight characterized by uniform thickening of the urothelium (simple hyperplasia) compared to the thickness of the control bladders. A small urinary calculus was noted within the lumen of one male rat given 2800 ppm. These bladder changes are consistent with chronic irritant effects on the urothelial lining of the bladder by urinary precipitates, crystals or calculi. There were no treatment-related microscopic bladder effects in Cohort 1A females given 2800 ppm biphenyl. All other histopathological observations in the liver, kidney and bladder as well as all other organs examined were spontaneous changes unassociated with the exposure to biphenyl.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Acoustic Startle Response (ASR) Habituation


There were no treatment-related effects on ASR in male or female Cohort 2A rats. The Treatment x Sex interaction was not significant (p = 0.7680), indicating that there was no statistically significant difference in ASR between males and females with treatment. This allowed the male and female data to be analyzed together to increase statistical power. There were no treatment-related effects on ASR in rats as indicated by the lack of a significant treatment main effect (p = 0.4661). The interaction of Treatment  Block was also not statistically significant (p = 0.0838), indicating that the habituation of the ASR was not affected by treatment.

Functional Observational Battery (FOB) Body Weights

There was no significant Treatment x Sex interaction (p = 0.5990), indicating that there was not a sex difference in FOB body weight response with treatment; therefore, males and females were analyzed together. The main effect of treatment was significant (p = 0.0470), indicating that biphenyl had a significant effect on body weight. However, comparisons of individual dose groups to the control group were not statistically-significant. Inspection of the data indicates that there was a slight decrease (approximately 5-6%) in FOB body weights (PND 63) of males and females of the 2800 ppm group that was deemed treatment-related based on similar results from the other F1 cohorts (see above). There were no treatment-related effects on FOB body weights of any other dose group.

Hand-Held and Open-Field Observations


Rectal Temperature

The Treatment x Sex interaction was not significant (p = 0.8606), indicating that there was no statistically significant difference in rectal temperature between males and females with treatment, which allowed both sexes to be analyzed together. There were no treatment-related effects on rectal temperature in male or female Cohort 2A rats (p = 0.3586).

Grip Performance

There were no treatment-related effects on grip performance. The Treatment x Sex interaction was not significant for either hindlimb (p = 0.1752) or forelimb (p = 0.6093) grip performance, indicating that there was no statistically significant difference in grip performance between males and females with treatment, which allowed both sexes to be analyzed together. There were no treatment-related effects on forelimb grip performance (p = 0.6592) in male or female Cohort 2A rats. However, the treatment main effect for hindlimb grip performance was statistically significant (p = 0.0137). Comparisons of individual dose groups to the control group were statistically-significant for the 2800 ppm dose group only. This statistically-identified difference was not considered treatment-related primarily due to the small magnitude of the change (11 or 14% decrease in males and females, respectively) considering the variability in this endpoint as demonstrated by coefficients of variation from 17 to 21% for control females and males, respectively. In addition, this slight decrease in hindlimb grip performance of the 2800 ppm group was not deemed treatment-related because:

1) There was no dose-related response. In the females, the treated groups had a flat dose response, while the control was slightly higher than the other groups. In the males, the 300 ppm dose group had an 11% increase in mean hindlimb grip performance, while the 2800 ppm dose group had an 11% decrease in mean hindlimb grip performance. This suggests the difference in the 2800 ppm groups was due to normal random variation in this behavioral measure.

2) There was no treatment-related effect in landing foot splay (see below). With decreased hindlimb grip performance one would expect an increase in hindlimb landing foot splay. The landing foot splay values of the 2800 ppm groups were similar to the controls in terms of absolute values.

3) There were no treatment-related FOB observations that would correlate with an effect on hindlimb grip performance (i.e., no effects on hindlimb extensor thrust response, muscle tone, or gait). This supports the lack of a treatment-related functional effect on the hindlimbs.

4) There were no neuropathological lesions in either the central or peripheral nervous system or the skeletal muscle that would support a structural or functional effect in the hindlimbs (see below).

Landing Foot Splay


The Treatment x Sex interaction was not significant (p = 0.5988), indicating that there was no statistically significant difference in landing foot splay between males and females with treatment, which allowed both sexes to be analyzed together. There were no treatment-related effects on landing foot splay in male or female Cohort 2A rats (p = 0.5665).

Motor Activity

There were no treatment-related effects on motor activity. The Treatment × Sex interaction was not significant (p = 0.2744), i.e., there was no statistically significant difference in motor activity across sexes with treatment. Furthermore, there was no statistically significant treatment main effect (p = 0.4275), i.e., with male and female data considered together, treatment did not affect motor activity. The Treatment × Interval interaction was also not statistically significant (p = 0.5844), which indicated that when male and female data were considered together, the distribution of motor activity counts within each session was not significantly affected by treatment. Together, these results indicate that biphenyl had no significant effect on motor activity in either males or females at any dose level tested.


Brain Weight and Gross Brain Measurements – Cohort 2A Offspring

There were no treatment-related changes in brain weights or gross brain measurements of males or females from any dose group.

Neuropathological Observations – Cohort 2A Offspring

There were no treatment-related histopathologic effects on the central or peripheral nervous system in males or females administered 2800 ppm biphenyl. In addition, microscopic examination of the brain and spinal cord from high-dose males and females that were stained with Fluoro-Jade did not reveal any treatment-related effects. All histopathological observations were interpreted to be spontaneous alterations, unassociated with dietary administration of biphenyl.

Brain Morphometrics – Cohort 2A Offspring

As part of the morphometric assessment, 3 structures in the cerebral cortex were measured in block #3, 4 structures in the thalamus and hippocampus were measured in block # 4, and 3 structures in the cerebellum are measured in block # 10.
There were no treatment-related effects on brain morphometrics in males or females administered 2800 ppm of biphenyl.
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

SRBC Antibody Response

The immunotoxic potential of biphenyl was assessed through the evaluation of the primary antibody response to sheep red blood cells (SRBCs) using an enzyme-linked immunosorbant assay (ELISA) approach that measured the concentration of serum anti-SRBC IgM. The SRBC antibody response is one of the few immunotoxicological endpoints that requires all of the cellular components of a classical immune response (e.g., B-cells, T-cells, macrophages) and thus, is a sensitive indicator of a chemical’s immunotoxic potential.

Analysis of the data did not identify a statistically significant difference between the biphenyl treatment groups and the vehicle control. The responses of three females in the 2800 ppm group were identified as statistical outliers; however, because the values were highly variable throughout the treatment group, these data were included in the analyses.

Analysis of the data for the positive control male and female groups revealed an expected response of statistically significant and greater than 99% reduction in the anti-SRBC IgM response.

Based on these results, biphenyl did not exhibit evidence of immunotoxicity at any dose level as it did not result in a treatment-related effect on the primary immune response to SRBCs in male and female rats.
The F1 generation has several different cohorts and they all collect the same basic data aside from any unique use they are evaluated for. There is no separate entry fields for this data so rather than greatly confuse and muddle the existing entry fields the authors stipulate that the the findings for all of the general toxicity parameters for the various F1 cohorts are essentially the same and there were no unique findings that would affect the stated No Effect Levels.
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
300 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
2 800 ppm
Sex:
male/female
Basis for effect level:
sexual maturation
developmental neurotoxicity
developmental immunotoxicity
Clinical signs:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Litter Observations – F2 Litters

Observations recorded in the offspring occurred at low frequency and did not show a dose-response relationship and therefore, were judged to have no relationship to treatment. One mid-dose litter contained one pup with anophthalmia and one high-dose litter contained one pup with an umbilical hernia
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Litter Size – P2/F2 Animals

Dietary exposure to biphenyl at doses up to 2800 ppm had no treatment-related effect on number of live pups born/litter or subsequent litter size measurements on LD 1, 4, 7, 14, or 21.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Pup Body Weights – F2 Offspring

There were no treatment-related effects on pup body weights at < 2800 ppm biphenyl during lactation. Although high-dose pup body weights on PND 7 were decreased relative to controls in the F1 generation, the pup body weight effect was not reproduced in the F2 generation. This may be in part due to a lesser effect on lactation feed consumption during the first week of lactation.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Animals were taken to weaning and sacrificed, so there is no FC data
Food efficiency:
not specified
Description (incidence and severity):
Animals were taken to weaning and sacrificed, so there is no FC data
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:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Nipple/Areolae Retention – F2 Offspring

There were no significant, treatment-related differences in nipple/areolae retention in either male or female offspring.

Anogenital Distance – F2 Offspring
There were no treatment-related effects on absolute or relative AGD in F2 males or females at any dose level. Although, there was a statistically identified decrease in relative AGD in F2 females at 300 and 2800 ppm, it was deemed spurious and unrelated to treatment as the difference was not dose related, there were no treatment-related effects on absolute AGD in F2 females or absolute or relative AGD in F2 males, and there were no treatment-related effects on absolute or relative AGD in males or female in the F1 offspring. Furthermore, lengthening of AGD is androgen-driven during development of the fetal rat (Foster et al., 2001; Scott et al., 2008). Therefore, endocrine-relevant effects on AGD in females would result in an increased value, indicative of androgenic activity (Hotchkiss et al., 2007, Wolf et al., 2002). As such, the slight decrease in relative female AGD in the biphenyl-treated groups is not considered toxicologically relevant.

Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Organ and Organ/Body Weights Summary – Non-selected F2 PND 22 Weanlings

There were no statistically identified or treatment-related changes in final body weights, brain, liver, kidneys, spleen, or thymus weights at any dose level. In addition, there were no effects on uterine weights in PND 22 unselected weanlings, indicating a lack of estrogenic response by biphenyl.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Gross Pathology Observations – Non-selected F2 PND 22 Weanlings

There were no treatment-related gross pathological observations attributed to biphenyl exposure in the non-selected PND 22 weanlings.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Description (incidence and severity):
Developmental immunotoxicity was not conducted on the F2
Developmental neurotoxicity was not conducted on the F2
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
2 800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
Critical effects observed:
no
Reproductive effects observed:
no

The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Thyroid Hormone Summary – (T3, T4, TSH) – PND 4 F1Culled Pups Pooled by Litter

Thyroid Hormone Summary – (T3, T4, TSH) – F1 Males – PND 22 (Unselected Weanlings) Thyroid Hormone Summary – (T3, T4, TSH) – F1 Females – PND 22 (Unselected Weanlings)
DOSE (PPM)   T3 T4 TSH Dose (PPM)   T3 T4 TSH DOSE (PPM)   T3 T4 TSH
    NG/DL UG/DL NG/ML     NG/DL UG/DL NG/ML     NG/DL UG/DL NG/ML
0 MEAN 77 1.81 1.5 0 MEAN 178.2 4.81 1.3 0 MEAN 163.5 4.24 1.1
S.D. 5.1 0.26 0.9 S.D. 18.7 0.73 0.6 S.D. 19.1 0.65 0.4
N= 10 10 10 N= 10 10 10 N= 10 10 10
300 MEAN 73 1.72 1.9 300 MEAN 172.5 4.84 2.1 300 MEAN 160.8 4.43 1.1
S.D. 3 0.18 1 S.D. 7.3 0.62 1.3 S.D. 15.3 0.59 0.4
N= 10 10 10 N= 10 10 10 N= 10 10 10
1000 MEAN 72.1* 1.95 1.2 1000 MEAN 162.8 4.94 1.1 1000 MEAN 173.2 4.63 1.5
S.D. 3.3 0.29 0.5 S.D. 18.1 0.78 0.4 S.D. 12.1 0.66 1
N= 10 10 10 N= 10 10 10 N= 10 10 10
2800 MEAN 72.1* 1.79 4.0* 2800 MEAN 169.6 4.71 1 2800 MEAN 161.1 4.87 1.4
S.D. 4.6 0.2 1.4 S.D. 11.4 0.49 0 S.D. 17.6 1.07 0.6
N= 10 10 10 N= 10 10 10 N= 10 10 10

Serum T3, T4, and TSH Levels in Male and Female PND 4 F2 Pups
Dose (ppm) Males and Females
T3 (ng/dL) % Change T4 (µg/dL) % Change TSH (ng/mL) % Change
0 70.3 NA 1.53 NA 3.8 NA
300 69.1 -1.7 1.5 -2 3 -21.1
1000 66.4 -5.5 1.47 -3.9 3.4 -10.5
2800 65.1* -7.4 1.39 -9.2 3.4 -10.5
Conclusions:
This biphenyl F1-extended one generation study established a systemic no-observed-adverse-effect level (NOAEL) of 1000 ppm (75 mg/kg/day) in males of both generations based on bladder and kidney toxicity, and 300 ppm (25 mg/kg/day) in females of both generations based on kidney toxicity. The NOAEL for reproduction, endocrine, developmental neurotoxicity and developmental immunotoxicity is the highest dose tested of 2800 ppm (215 mg/kg/day) biphenyl as there were no indications of these effects in either the P1 or Cohort 1-3 offspring. Overall, biphenyl-treatment-related effects and target organs were consistent with its previous toxicity dataset and animals exposed during critical windows of development did not display any unique susceptibility to biphenyl-induced toxicity.
Executive summary:

The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 used in this form. The laboratory nomenclature is retained to be consistent with the study report.

The purpose of this one-generation reproduction dietary toxicity study was to evaluate the potential effects of biphenyl on male and female rat reproductive function, as well as potential effects on numerous endpoints in the F1 offspring, including survival, growth, and development of the nervous, immune and reproductive systems.  In addition, endocrine and selected systemic toxicity parameters were assessed.

Overall conclusions from this study were as follows (with more detail provided below):

•   This biphenyl extended one-generation study identified kidney, urinary bladder and liver as target organs for biphenyl-induced toxicity, which was consistent with its previous toxicity dataset.

•   The systemic no-observed-adverse-effect-levels (NOAELs) were the same in both generations with the NOAEL of 1000 ppm

(75 mg/kg/day) for males based on bladder and kidney toxicity, and the NOAEL of 300 ppm (25 mg/kg/day) for females based on kidney toxicity.

•   There was no evidence of treatment-related reproductive toxicity, including an absence of effects on reproductive and litter parameters in both generations up to the highest dose tested (NOAEL of

2800 ppm or 215 mg/kg/day).

•   Although high-dose pup body weights on PND 7 were slightly decreased relative to controls in the F1 generation, the pup body weight effect appeared to be secondary to decreased maternal feed consumption and this effect was not reproduced in the F2 generation.

•   There were no effects on the estrogen-, androgen-, or thyroid-related endocrine pathways at any dose of biphenyl.

•   For developmental neurotoxicity, there were no treatment-related effects on neurobehavior or neuropathology at any dose levels in either Cohort 2A or 2B animals.

•   Biphenyl exposure did not result in developmental immunotoxicity at any dose level.

Groups of 26 male and 26 female young adult rats were fed control or biphenyl- containing diets supplying 0, 300, 1000, and 2800 ppm biphenyl (~25, 75, and 215 mg biphenyl/kg/day, respectively, the nominal doses) for approximately ten weeks prior to breeding, and continuing through breeding (two weeks).  An additional satellite group of P1 females (4/dose) was included primarily for assessments of kidney function in adult non-pregnant females during the pre-breeding period.  The satellite female group was given biphenyl-containing diets concurrent with the P1 animals during pre-breeding. Satellite females were removed from study/terminated at the end of the pre-breeding period.  After breeding, P1 males continued on the test diets for an additional 7-8 weeks

(19-20 weeks total exposure).  After breeding, P1 females continued on the test diets through gestation and lactation (16-18 weeks total exposure).  In-life parameters included clinical observations/detailed clinical observations, feed consumption, body weights, estrous cycle evaluation, litter and fertility data, thyroid hormone measurement, clinical chemistry/hematology parameters, and urinalysis.  In addition, post-mortem evaluations of P1 adults included gross pathology, organ weights, histopathology, and an evaluation of sperm parameters.

In-life parameters evaluated in all F1 offspring included clinical observations/detailed clinical observations, body weights, feed consumption, anogenital distance, nipple retention, and puberty onset.

F1 offspring were divided into Cohorts 1A, 1B, 2A, 2B and 3 at weaning (postnatal day (PND) 21) as follows:

•   Cohort 1A (22-24/sex/dose, 1 pup/sex/litter) were used to evaluate reproductive/endocrine toxicity, which included estrous cycle evaluation and post-mortem evaluations focused on reproductive organs, sperm assessment, and ovarian follicle counts on PND 90.  This group also was used to assess general systemic and thyroid toxicity, which included clinical chemistry/hematology parameters, thyroid hormone assessment, and urinalysis.  Post-mortem evaluations in Cohort 1A (PND 90) also included gross pathology, organ weights, and histopathology on a wide range of tissues, including thyroids.

•   Cohort 1B animals (21-24/sex/dose, 1 pup/sex/litter) were known as the endocrine group and designated to clarify any equivocal responses seen in the Cohort 1A animals.  This group was used to generate a second generation of offspring, based on equivocal incidences of dystocia in the P1 animals.  Additional in-life parameters evaluated in the Cohort 1B P2 animals included an estrous cycle evaluation, litter and fertility data, thyroid hormone measurements, clinical chemistry/hematology parameters, and urinalysis.  Post-mortem evaluations in Cohort 1B P2 animals occurred in males after approximately 19 weeks of postnatal exposure (PND 138-148) and in females on LD 22 (after approximately 21 weeks of postnatal exposure).  Evaluations included sperm assessment, gross pathology, organ weights, and histopathology with a primary focus on tissues affected in Cohort 1A, including kidney, liver and urinary bladder.

•   The Cohort 2A and 2B animals (21-22/sex/dose, 1 pup/sex/litter) were used to assess potential developmental neurotoxicity (DNT) as follows:

o Cohort 2A (11-12/sex/dose, 1 pup/sex/litter) were used for developmental neurotoxicity (DNT) assessments, which included functional observational battery (FOB), motor activity, and acoustic startle response (ASR).  On PND 78, Cohort 2A F1 animals were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation and brain morphometry.

o Cohort 2B (10/sex/dose, 1 pup/litter) underwent necropsy on PND 22, which included brain weight collection in these weanlings and immersion fixation of tissues for examination of neuropathology.

•   Cohort 3 (10/sex/dose, 1 pup/litter) were used for developmental immunotoxicity (DIT) assessments through the evaluation of the primary antibody response to sheep red blood cells (SRBCs).

Additional data were gathered from F1 offspring not assigned to the Cohorts above. Weanlings not assigned to Cohorts 1-3 were considered “unselected” weanlings.  On PND 22, these unselected weanlings were euthanized for assessment of systemic toxicity, which included thyroid hormone assessment, selected organ weights, and post-mortem examinations (gross pathology and histopathology) in 10/sex/dose.  In addition, selected pups culled on PND 4 were used to assess thyroid hormone levels and histopathology.

Nominal dose levels (mg/kg/day) were achieved across both P1 and F1 generations; however, during some study intervals (e.g., PND 35-56), F1 offspring had greater biphenyl intake per kg body weight than P1 males and pre-breeding females.  F1 offspring diets, which were adjusted to one-half normal concentrations from PND 21-35, returned to full concentration diets on PND 35; however, offspring continued to eat more diet/kg body weight, resulting in higher dose levels than the adults.

Systemic toxicity was assessed across life stages.  In the parental generation, high-dose biphenyl caused slight decreases in body weights and body weight gains, corresponding to decreased feed consumption in males and females throughout pre-breeding and continuing post-breeding for males and through the first week of gestation and then in the lactation period for P1 females.  By PND 7, there was a treatment-related decrease in high-dose male and female F1 pup weights (6.8-8.2%) compared to the control group. This period of lactation (LD 0-7) corresponded with the period of highest test material intake by the P1 dams.  In addition, the effects on pup weight were consistent with the lower maternal body weights on LD 1 (4.5%), 4 (5.2%), and 7 (6.4%) and treatment- related decreases in maternal feed consumption during the first week of lactation, which potentially lowered the ability of the dams to maintain offspring body weights.

In the offspring Cohorts 1A, 1B, and 2A, body weights, body weight gains and feed consumption were decreased in the high dose group throughout the majority of the exposure period between PND 42 and the respective cohort termination.  In Cohort 1B P2 females, body weights were decreased throughout gestation by ≤ 6.7% relative to controls with a corresponding decrease in GD 0-7 body weight gain of 17% and a slight decrease in feed consumption compared to controls.  Cohort 1B P2 lactation body weights (6.0-8.3%), body weight gains (33.3-59.7%) and feed consumption (2.4-8.6%) were decreased during the first week of lactation, which subsequently increased during the last two weeks of lactation.  There were no treatment-related effects on F2 pup body weights at < 2800 ppm biphenyl during lactation.  Although high-dose F2 pup body weights on PND 7 were decreased relative to controls in the F1 generation, the pup body weight effect was not reproduced in the F2 generation.

There were no treatment-related changes in any of the hematology or clinical chemistry parameters in males or females at any dose level across the two generations.  Urinalysis of non-pregnant satellite group females given 2800 ppm revealed the presence of treatment-related smooth ovoid crystals of various sizes (10-40 microns) and shapes in the urinary sediment.  In Cohort 1A females and Cohort 1B P2 males given 2800 ppm, there was a treatment-related increase in urine volume and decrease in specific gravity. Urinary sediments from Cohort 1A males and females, and Cohort 1B P2 males given

2800 ppm had treatment-related presence of amorphous phosphate crystals.  Cohort 1B P2 males and P2 females given 2800 ppm had treatment-related, higher severity of hematuria (blood in the urine) compared to controls.

This biphenyl extended one-generation study identified kidney, urinary bladder and liver as target organs for biphenyl-induced toxicity.  Treatment-related organ weight changes were confined to the liver and kidney in the P2 generation.  Cohort 1B P2 males given

1000 or 2800 ppm and females given 2800 ppm had treatment-related higher absolute

and relative liver weights and Cohort 1B P2 females given 2800 ppm had higher absolute and relative kidney weights compared to the control.

With the exception of treatment-related gross findings of calculi in the urinary bladder and thickened bladder wall in 2 of 19 Cohort 1B P2 males given 2800 ppm, there were no other gross pathological observations in any of the organs examined in males or females at any dose level across the two generations. Within the 2800 ppm group across the two generations, a small proportion of males had treatment-related histopathological changes in the kidney largely confined to the renal papilla, renal pelvic epithelium and/or the collecting ducts.  Salient treatment-related changes include focal or multifocal hyperplasia of epithelium of the papilla and/or the renal pelvic epithelium, multifocal epithelial hyperplasia and hypertrophy of the collecting ducts, and multifocal subacute to chronic inflammation of the papilla and/or pelvic epithelium.  The severity of most of these changes were very slight or slight or occasionally moderate.  Other less frequent treatment-related changes variably noted between P1, Cohort 1A and P2 males given 2800 ppm included slight multifocal necrosis of the collecting duct epithelium, slight focal edema at the tip of the papilla, slight focal ulceration of the papilla, and slight hemorrhage in the renal pelvis.  A few of the high- dose males, particularly in the Cohort 1A and P2 generations had treatment-related presence of an eosinophilic to red, fine granular to aggregated precipitated material in the renal pelvis admixed with red blood cells consistent with calculi, which were likely urinary precipitates of the test material and/or its metabolites.  The treatment-related changes in the kidney of males given 2800 ppm are consistent with chronic irritant effects of these urinary precipitates or calculi on the collecting ducts, renal pelvis and papilla.

P1 females given 1000 or 2800 ppm had treatment related very slight or slight degeneration of tubules largely confined to the outer stripe of the outer medulla.  The change was characterized by very slightly dilated tubular lumens at multiple foci, lined by epithelial cells that variably contained fine cytoplasmic vacuoles and were very slightly reduced in cell height (attenuated) compared to the controls.  The lumens of these tubules often contained increased amounts of eosinophilic homogeneous or globular material compared to the controls.  In addition, Cohort 1A and P2 females given 2800 ppm had treatment-related increased incidence and severity of medullary tubular mineralization.  Other treatment-related histopathologic changes noted in a small proportion of 2800 ppm P2 females included slight focal or multifocal hyperplasia of papillary epithelium and slight multifocal hyperplasia and hypertrophy of the epithelium of the collecting ducts in the papilla.

The urinary bladder of P1, Cohort 1A and P2 males and females given 2800 ppm had treatment-related, very slight or slight simple diffuse urothelial hyperplasia and a very slight multifocal subacute to chronic inflammation in the lamina propria underlying the urothelial lining of the bladder.  The hyperplasia was characterized by uniform thickening of the urothelium (simple hyperplasia).  These changes were consistent with chronic irritant effects on the urothelial lining of the bladder by urinary precipitates or calculi

related to the test material.

In the liver, P1 and Cohort 1A males given 1000 or 2800 ppm, and P1 and Cohort 1A

females given 2800 ppm had very slight hypertrophy of centrilobular/midzonal hepatocytes with increased cytoplasmic eosinophilia.  In the second generation, P2 males and females given 1000 or 2800 ppm had a very slight or slight treatment-related centrilobular/midzonal hypertrophy with increased cytoplasmic eosinophilia.  Based on the absence of other treatment-related histopathological findings such as necrosis, inflammation, fibrosis, vacuolation, proliferation, degeneration, etc., in the affected livers, or any clinical chemistry changes indicative of liver injury, the very slight or slight hepatocyte hypertrophy with increased cytoplasmic eosinophilia was interpreted to be an adaptive, non-adverse change in response to the continued ingestion of biphenyl.

There was no evidence of treatment-related reproductive toxicity or effects on the estrogen-, androgen-, or thyroid-related endocrine pathways at any dose of biphenyl. There were two incidences of dystocia in each of the low and mid dose P1 dams. Although there were no incidences of dystocia in the P1 high dose, there were no previous incidences of dystocia in the laboratory historical control data and therefore interpretation of this rare occurrence was deemed equivocal based on the P1 data. Therefore, a second generation was produced to help clarify the interpretation of this finding.  No incidences of dystocia occurred in the P2 biphenyl-treated dams, and one incidence of dystocia occurred in one P2 control dam.  Based on these results in the P2 generation, the dystocia seen in the P1 litters was considered spurious and unrelated to treatment.  The transient body weight effect in the high-dose F1 offspring, which was not reproduced in the F2 offspring, was attributed to decreased maternal feed consumption during early lactation and not a direct effect of biphenyl on pup growth.

For developmental neurotoxicity, there were no treatment-related effects on neuropathology at any dose levels in either Cohort 2A or 2B animals.  There were no effects on neurobehavioral endpoints at any dose of biphenyl, despite exposures during critical windows of development.

Biphenyl exposure did not result in developmental immunotoxicity at any dose level, as evidenced by lack of a difference from control in SRBC antibody response in Cohort 3 treated animals.

There were no treatment-related effects in the F2 generation offspring at any dose level. There were no treatment-related effects on litter observations, litter size, pup weights, anogenital distance, nipple retention, thyroid hormone levels, organ weights, gross pathology and thyroid gland histopathology.

This biphenyl F1-extended one generation study established a systemic no-observed-

adverse-effect level (NOAEL) of 1000 ppm (75 mg/kg/day) in males of both generations based on bladder and kidney toxicity, and 300 ppm (25 mg/kg/day) in females of both generations based on kidney toxicity.  The NOAEL for reproduction, endocrine, developmental neurotoxicity and developmental immunotoxicity is the highest dose tested of 2800 ppm (215 mg/kg/day) biphenyl as there were no indications of these effects in either the parental animals or the offspring across both generations.  Overall, biphenyl-treatment-related effects and target organs were consistent with its previous toxicity dataset and animals exposed during critical windows of development did not display any unique susceptibility to biphenyl-induced toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
350 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
acceptable
Additional information

The testing laboratory for this study uses "P1 and P2" for their nomenclature rather than the P0 and P1 used in this form. The laboratory nomenclature is retained to be consistent with the study report.

The purpose of this one-generation reproduction dietary toxicity study was to evaluate the potential effects of biphenyl on male and female rat reproductive function, as well as potential effects on numerous endpoints in the F1 offspring, including survival, growth, and development of the nervous, immune and reproductive systems.  In addition, endocrine and selected systemic toxicity parameters were assessed.

Overall conclusions from this study were as follows (with more detail provided below):

•   This biphenyl extended one-generation study identified kidney, urinary bladder and liver as target organs for biphenyl-induced toxicity, which was consistent with its previous toxicity dataset.

•   The systemic no-observed-adverse-effect-levels (NOAELs) were the same in both generations with the NOAEL of 1000 ppm

(75 mg/kg/day) for males based on bladder and kidney toxicity, and the NOAEL of 300 ppm (25 mg/kg/day) for females based on kidney toxicity.

•   There was no evidence of treatment-related reproductive toxicity, including an absence of effects on reproductive and litter parameters in both generations up to the highest dose tested (NOAEL of

2800 ppm or 215 mg/kg/day).

•   Although high-dose pup body weights on PND 7 were slightly decreased relative to controls in the F1 generation, the pup body weight effect appeared to be secondary to decreased maternal feed consumption and this effect was not reproduced in the F2 generation.

•   There were no effects on the estrogen-, androgen-, or thyroid-related endocrine pathways at any dose of biphenyl.

•   For developmental neurotoxicity, there were no treatment-related effects on neurobehavior or neuropathology at any dose levels in either Cohort 2A or 2B animals.

•   Biphenyl exposure did not result in developmental immunotoxicity at any dose level.

Groups of 26 male and 26 female young adult rats were fed control or biphenyl- containing diets supplying 0, 300, 1000, and 2800 ppm biphenyl (~25, 75, and 215 mg biphenyl/kg/day, respectively, the nominal doses) for approximately ten weeks prior to breeding, and continuing through breeding (two weeks).  An additional satellite group of P1 females (4/dose) was included primarily for assessments of kidney function in adult non-pregnant females during the pre-breeding period.  The satellite female group was given biphenyl-containing diets concurrent with the P1 animals during pre-breeding. Satellite females were removed from study/terminated at the end of the pre-breeding period.  After breeding, P1 males continued on the test diets for an additional 7-8 weeks

(19-20 weeks total exposure).  After breeding, P1 females continued on the test diets through gestation and lactation (16-18 weeks total exposure).  In-life parameters included clinical observations/detailed clinical observations, feed consumption, body weights, estrous cycle evaluation, litter and fertility data, thyroid hormone measurement, clinical chemistry/hematology parameters, and urinalysis.  In addition, post-mortem evaluations of P1 adults included gross pathology, organ weights, histopathology, and an evaluation of sperm parameters.

In-life parameters evaluated in all F1 offspring included clinical observations/detailed clinical observations, body weights, feed consumption, anogenital distance, nipple retention, and puberty onset.

F1 offspring were divided into Cohorts 1A, 1B, 2A, 2B and 3 at weaning (postnatal day (PND) 21) as follows:

•   Cohort 1A (22-24/sex/dose, 1 pup/sex/litter) were used to evaluate reproductive/endocrine toxicity, which included estrous cycle evaluation and post-mortem evaluations focused on reproductive organs, sperm assessment, and ovarian follicle counts on PND 90.  This group also was used to assess general systemic and thyroid toxicity, which included clinical chemistry/hematology parameters, thyroid hormone assessment, and urinalysis.  Post-mortem evaluations in Cohort 1A (PND 90) also included gross pathology, organ weights, and histopathology on a wide range of tissues, including thyroids.

•   Cohort 1B animals (21-24/sex/dose, 1 pup/sex/litter) were known as the endocrine group and designated to clarify any equivocal responses seen in the Cohort 1A animals.  This group was used to generate a second generation of offspring, based on equivocal incidences of dystocia in the P1 animals.  Additional in-life parameters evaluated in the Cohort 1B P2 animals included an estrous cycle evaluation, litter and fertility data, thyroid hormone measurements, clinical chemistry/hematology parameters, and urinalysis.  Post-mortem evaluations in Cohort 1B P2 animals occurred in males after approximately 19 weeks of postnatal exposure (PND 138-148) and in females on LD 22 (after approximately 21 weeks of postnatal exposure).  Evaluations included sperm assessment, gross pathology, organ weights, and histopathology with a primary focus on tissues affected in Cohort 1A, including kidney, liver and urinary bladder.

•   The Cohort 2A and 2B animals (21-22/sex/dose, 1 pup/sex/litter) were used to assess potential developmental neurotoxicity (DNT) as follows:

o Cohort 2A (11-12/sex/dose, 1 pup/sex/litter) were used for developmental neurotoxicity (DNT) assessments, which included functional observational battery (FOB), motor activity, and acoustic startle response (ASR).  On PND 78, Cohort 2A F1 animals were perfused for central nervous system (CNS) and peripheral nerve neuropathology evaluation and brain morphometry.

o Cohort 2B (10/sex/dose, 1 pup/litter) underwent necropsy on PND 22, which included brain weight collection in these weanlings and immersion fixation of tissues for examination of neuropathology.

•   Cohort 3 (10/sex/dose, 1 pup/litter) were used for developmental immunotoxicity (DIT) assessments through the evaluation of the primary antibody response to sheep red blood cells (SRBCs).

Additional data were gathered from F1 offspring not assigned to the Cohorts above. Weanlings not assigned to Cohorts 1-3 were considered “unselected” weanlings.  On PND 22, these unselected weanlings were euthanized for assessment of systemic toxicity, which included thyroid hormone assessment, selected organ weights, and post-mortem examinations (gross pathology and histopathology) in 10/sex/dose.  In addition, selected pups culled on PND 4 were used to assess thyroid hormone levels and histopathology.

Nominal dose levels (mg/kg/day) were achieved across both P1 and F1 generations; however, during some study intervals (e.g., PND 35-56), F1 offspring had greater biphenyl intake per kg body weight than P1 males and pre-breeding females.  F1 offspring diets, which were adjusted to one-half normal concentrations from PND 21-35, returned to full concentration diets on PND 35; however, offspring continued to eat more diet/kg body weight, resulting in higher dose levels than the adults.

Systemic toxicity was assessed across life stages.  In the parental generation, high-dose biphenyl caused slight decreases in body weights and body weight gains, corresponding to decreased feed consumption in males and females throughout pre-breeding and continuing post-breeding for males and through the first week of gestation and then in the lactation period for P1 females.  By PND 7, there was a treatment-related decrease in high-dose male and female F1 pup weights (6.8-8.2%) compared to the control group. This period of lactation (LD 0-7) corresponded with the period of highest test material intake by the P1 dams.  In addition, the effects on pup weight were consistent with the lower maternal body weights on LD 1 (4.5%), 4 (5.2%), and 7 (6.4%) and treatment- related decreases in maternal feed consumption during the first week of lactation, which potentially lowered the ability of the dams to maintain offspring body weights.

In the offspring Cohorts 1A, 1B, and 2A, body weights, body weight gains and feed consumption were decreased in the high dose group throughout the majority of the exposure period between PND 42 and the respective cohort termination.  In Cohort 1B P2 females, body weights were decreased throughout gestation by ≤ 6.7% relative to controls with a corresponding decrease in GD 0-7 body weight gain of 17% and a slight decrease in feed consumption compared to controls.  Cohort 1B P2 lactation body weights (6.0-8.3%), body weight gains (33.3-59.7%) and feed consumption (2.4-8.6%) were decreased during the first week of lactation, which subsequently increased during the last two weeks of lactation.  There were no treatment-related effects on F2 pup body weights at < 2800 ppm biphenyl during lactation.  Although high-dose F2 pup body weights on PND 7 were decreased relative to controls in the F1 generation, the pup body weight effect was not reproduced in the F2 generation.

There were no treatment-related changes in any of the hematology or clinical chemistry parameters in males or females at any dose level across the two generations.  Urinalysis of non-pregnant satellite group females given 2800 ppm revealed the presence of treatment-related smooth ovoid crystals of various sizes (10-40 microns) and shapes in the urinary sediment.  In Cohort 1A females and Cohort 1B P2 males given 2800 ppm, there was a treatment-related increase in urine volume and decrease in specific gravity. Urinary sediments from Cohort 1A males and females, and Cohort 1B P2 males given

2800 ppm had treatment-related presence of amorphous phosphate crystals.  Cohort 1B P2 males and P2 females given 2800 ppm had treatment-related, higher severity of hematuria (blood in the urine) compared to controls.

This biphenyl extended one-generation study identified kidney, urinary bladder and liver as target organs for biphenyl-induced toxicity.  Treatment-related organ weight changes were confined to the liver and kidney in the P2 generation.  Cohort 1B P2 males given

1000 or 2800 ppm and females given 2800 ppm had treatment-related higher absolute

and relative liver weights and Cohort 1B P2 females given 2800 ppm had higher absolute and relative kidney weights compared to the control.

With the exception of treatment-related gross findings of calculi in the urinary bladder and thickened bladder wall in 2 of 19 Cohort 1B P2 males given 2800 ppm, there were no other gross pathological observations in any of the organs examined in males or females at any dose level across the two generations. Within the 2800 ppm group across the two generations, a small proportion of males had treatment-related histopathological changes in the kidney largely confined to the renal papilla, renal pelvic epithelium and/or the collecting ducts.  Salient treatment-related changes include focal or multifocal hyperplasia of epithelium of the papilla and/or the renal pelvic epithelium, multifocal epithelial hyperplasia and hypertrophy of the collecting ducts, and multifocal subacute to chronic inflammation of the papilla and/or pelvic epithelium.  The severity of most of these changes were very slight or slight or occasionally moderate.  Other less frequent treatment-related changes variably noted between P1, Cohort 1A and P2 males given 2800 ppm included slight multifocal necrosis of the collecting duct epithelium, slight focal edema at the tip of the papilla, slight focal ulceration of the papilla, and slight hemorrhage in the renal pelvis.  A few of the high- dose males, particularly in the Cohort 1A and P2 generations had treatment-related presence of an eosinophilic to red, fine granular to aggregated precipitated material in the renal pelvis admixed with red blood cells consistent with calculi, which were likely urinary precipitates of the test material and/or its metabolites.  The treatment-related changes in the kidney of males given 2800 ppm are consistent with chronic irritant effects of these urinary precipitates or calculi on the collecting ducts, renal pelvis and papilla.

P1 females given 1000 or 2800 ppm had treatment related very slight or slight degeneration of tubules largely confined to the outer stripe of the outer medulla.  The change was characterized by very slightly dilated tubular lumens at multiple foci, lined by epithelial cells that variably contained fine cytoplasmic vacuoles and were very slightly reduced in cell height (attenuated) compared to the controls.  The lumens of these tubules often contained increased amounts of eosinophilic homogeneous or globular material compared to the controls.  In addition, Cohort 1A and P2 females given 2800 ppm had treatment-related increased incidence and severity of medullary tubular mineralization.  Other treatment-related histopathologic changes noted in a small proportion of 2800 ppm P2 females included slight focal or multifocal hyperplasia of papillary epithelium and slight multifocal hyperplasia and hypertrophy of the epithelium of the collecting ducts in the papilla.

The urinary bladder of P1, Cohort 1A and P2 males and females given 2800 ppm had treatment-related, very slight or slight simple diffuse urothelial hyperplasia and a very slight multifocal subacute to chronic inflammation in the lamina propria underlying the urothelial lining of the bladder.  The hyperplasia was characterized by uniform thickening of the urothelium (simple hyperplasia).  These changes were consistent with chronic irritant effects on the urothelial lining of the bladder by urinary precipitates or calculi

related to the test material.

In the liver, P1 and Cohort 1A males given 1000 or 2800 ppm, and P1 and Cohort 1A

females given 2800 ppm had very slight hypertrophy of centrilobular/midzonal hepatocytes with increased cytoplasmic eosinophilia.  In the second generation, P2 males and females given 1000 or 2800 ppm had a very slight or slight treatment-related centrilobular/midzonal hypertrophy with increased cytoplasmic eosinophilia.  Based on the absence of other treatment-related histopathological findings such as necrosis, inflammation, fibrosis, vacuolation, proliferation, degeneration, etc., in the affected livers, or any clinical chemistry changes indicative of liver injury, the very slight or slight hepatocyte hypertrophy with increased cytoplasmic eosinophilia was interpreted to be an adaptive, non-adverse change in response to the continued ingestion of biphenyl.

There was no evidence of treatment-related reproductive toxicity or effects on the estrogen-, androgen-, or thyroid-related endocrine pathways at any dose of biphenyl. There were two incidences of dystocia in each of the low and mid dose P1 dams. Although there were no incidences of dystocia in the P1 high dose, there were no previous incidences of dystocia in the laboratory historical control data and therefore interpretation of this rare occurrence was deemed equivocal based on the P1 data. Therefore, a second generation was produced to help clarify the interpretation of this finding.  No incidences of dystocia occurred in the P2 biphenyl-treated dams, and one incidence of dystocia occurred in one P2 control dam.  Based on these results in the P2 generation, the dystocia seen in the P1 litters was considered spurious and unrelated to treatment.  The transient body weight effect in the high-dose F1 offspring, which was not reproduced in the F2 offspring, was attributed to decreased maternal feed consumption during early lactation and not a direct effect of biphenyl on pup growth.

For developmental neurotoxicity, there were no treatment-related effects on neuropathology at any dose levels in either Cohort 2A or 2B animals.  There were no effects on neurobehavioral endpoints at any dose of biphenyl, despite exposures during critical windows of development.

Biphenyl exposure did not result in developmental immunotoxicity at any dose level, as evidenced by lack of a difference from control in SRBC antibody response in Cohort 3 treated animals.

There were no treatment-related effects in the F2 generation offspring at any dose level. There were no treatment-related effects on litter observations, litter size, pup weights, anogenital distance, nipple retention, thyroid hormone levels, organ weights, gross pathology and thyroid gland histopathology.

This biphenyl F1-extended one generation study established a systemic no-observed-

adverse-effect level (NOAEL) of 1000 ppm (75 mg/kg/day) in males of both generations based on bladder and kidney toxicity, and 300 ppm (25 mg/kg/day) in females of both generations based on kidney toxicity.  The NOAEL for reproduction, endocrine, developmental neurotoxicity and developmental immunotoxicity is the highest dose tested of 2800 ppm (215 mg/kg/day) biphenyl as there were no indications of these effects in either the parental animals or the offspring across both generations.  Overall, biphenyl-treatment-related effects and target organs were consistent with its previous toxicity dataset and animals exposed during critical windows of development did not display any unique susceptibility to biphenyl-induced toxicity.

Effects on developmental toxicity

Description of key information

Developmental toxicity studies have been conducted in rats and rabbits with no developmental toxicity findings.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was performed similar to OECD 414 with acceptable deviations. Sufficient information on methods and results was provided to evaluate data.
Remarks:
none
Justification for type of information:
none
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
: Limited or no information on environmental conditions and animals. Dams dosed from days 6-15 of gestation. Maternal mortality exceeded 10% at the highest dose. The dosing volume exceeded recommended volume. Limited information on clinical observations.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Woodlyn Farms, Guelph, Ontario, Canada
- Age at study initiation: no data
- Weight at study initiation: 175-200 g (females)
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS: no data
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: no details

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 12.5, 25, 50 and 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not applicable
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not indicated
- Length of cohabitation: not indicated
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Not indicated
- Further matings after two unsuccessful attempts: not indicated
- Verification of same strain and source of both sexes: not indicated
- Proof of pregnancy: positive vaginal smear referred to as day 1 of pregnancy
- Any other deviations from standard protocol: administration of test substance from implantation (day 6) through day 15 of gestation (rather than to the day prior to scheduled caesarean section)
Duration of treatment / exposure:
Day 6 through day 15 of gestation
Frequency of treatment:
Once daily
Duration of test:
Until day 22 of gestation
No. of animals per sex per dose:
18 to 20 mated females
Control animals:
yes
Details on study design:
No further details
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes: mortality

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: day 1, 6 through 15, and 22 of pregnancy

POST-MORTEM EXAMINATIONS: Yes : necropsy
- Sacrifice on gestation day 22
- Organs examined: no data
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one-thirds per litter
- Skeletal examinations: Yes: two-thirds per litter
- Head examinations: Yes: No data
Statistics:
Values of t for test group mean maternal body weight versus control group (Dunn's test)
Mean fetal parameters: proportion of a litter having a particular attribute calcuilated, mean and SE derived (Hald method)
Significant differences if p<0.05.
Indices:
Number of rats pregnant/number mated
Number of corpora lutea per pregnancy
Number of live fetuses per pregnancy
Dead and resorbed fetuses (%), i.e. (number of resorption sites + dead fetuses / total implants)x 100
Number of anomalous fetuses/number examined
Number of anomalous litters/number examined
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
1000 mg/kg bw/day dose group:
- resorption occurred in one litter
- 5 animals were found not to be pregnant (may be due to interference with implantation)
- in 5 pregnant dams mortality occurred. Each death occurred during the dosing period and was preceded by a sharp reduction in body weight and diarrhea.
125, 250 and 500 mg/kg bw/day dose groups:
- no signs of toxicity occurred.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Although 1000 mg/kg bw/day biphenyl was lethal for five dams, for the dams that survived, it did not affect the incidence of corpora lutea, live fetuses, or dead fetuses plus resorption sites, nor did it affect fetal weight. Although the latter was reduced, and the incidence of dead fetuses plus resorptions increased, these values were not statistically significant from the control animals. In both the 1000 and 500 mg/kg fetuses with missing and unossified sternebrae not statistically significant. The incidence and types of anomalies and ossifications in the biphenyl dosed groups and the control were also similar.
Results are presented for doses 0, 125, 250, 500 and 1000 mg/kg respectively:
- number of rats with live fetuses/number mated: 16/18, 20/20, 18/19, 18/20, 9/20
- number of corpora lutea per pregnancy (mean +/- SE): 12.6 +/-0.4, 12.9 +/-0.4, 13.7 +/-0.5, 13.3 +/-0.4, 12.5 +/-0.7
- number of live fetuses per pregnancy (mean +/- SE): 11.3 +/-0.7, 11.8 +/-0.6, 11.9 +/-0.6, 11.2 +/-0.5, 10.7 +/-1.3
- dead and resorbed fetuses (%): 4.8, 3.3, 6.1, 7.8, 13.7
- fetal weight (g mean +/- SE): 5.1 +/-0.1, 5.3 +/-0.1, 5.2 +/-0.1, 5.2 +/-0.1, 4.5 +/-0.3
- number of anomalous fetuses/number examined: 17/176, 22/236, 22/213, 35/199, 25/107
- number of anomalous litters/number examined: 8/16, 11/20, 13/18, 15/18, 6/9
Anomalies (number of fetuses affected):
- wavy ribs uni- and bilateral: 3, 7, 9, 8, 5
- extra ribs uni- and bilateral: 9, 12, 9, 15, 6
- 13th rib small sized: 1, 1, 2, 1, 0
- missing or unossified sternebrae: 4, 3, 4, 16, 17
- delayed ossification calvarium: 0, 2, 0, 0, 8
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No changes at this dose level
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Mortality occured during the dosing period in 10% of dams dosed with 1000 mg/kg bw. No statistically significant effects on fetotoxicity or teratogenicty occured at any of the doses evaluated.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 27, 2019 to November 1, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Test Material Name Biphenyl
Chemical Name 1,1’-Biphenyl
Synonyms None
Lot/Reference/Batch Number 8J026
Supplier Eastman Chemical

Purity/Characterization (Method of Analysis and Reference)
The purity of the test material was determined to be 99.88% (corrected for water) by gas chromatography with identification by gas chromatography mass spectrometry and Fourier transform infrared spectroscopy (Palumbo, 2019).

Test Material Stability Under Storage Conditions
The test material biphenyl, Lot 8J026, was not tested for neat test material stability. However, neat test material stability was conducted previously for biphenyl, Lot 5M009, under GLP conditions, determining the neat test material to be stable for 24 months under ambient storage conditions
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Species and Sex: Rabbits, time-mated females
Strain and Justification: New Zealand White (NZW) rabbits were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data, their use in previous studies, and the reliability of the commercial supplier.

Supplier and Location: Covance Research Products, Inc. (CRP), Greenfield, Indiana.
Age and Weight at Study Start: Sexually mature, time-mated female adult rabbits weighing 2800-3200 g.
Health Status and Acclimation: Upon arrival, all animals were acclimated to the laboratory for at least five days prior to the start of test material administration. During the acclimation period, each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
Housing

Animals were housed one per cage in stainless steel and plastic cages in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Cages had perforated plastic floors and were suspended above catch pans with absorbent non-contact bedding. Cages contained a J-type feeder and a pressure activated lixit valve-type watering system.

Animals were acclimated at Covance to Teklad Certified Global 2030C Rabbit Diet (Envigo, Madison, Wisconsin) with 0.5% apple flavoring for at least five days prior to breeding and shipment. A stepwise increase in an amount of daily feed allotted was implemented to aid in avoiding gastrointestinal disturbances during the acclimation period. Upon receipt, rabbits received approximately 75 g of Teklad Certified Global 2030C Rabbit Diet (Envigo, Madison, Wisconsin) with 0.5% apple flavoring in pelleted form. The amount of feed was increased up to approximately 150 g the following day until GD 6, and then control and respective formulated dietary concentrations of biphenyl were given from GD 7-28. Analyses of the feed were performed by Envigo, Teklad Diets to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants.
Municipal water was provided ad libitum. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants identified that would have interfered with the conduct or interpretation of the study. Copies of these analyses were maintained in the study file.


The following environmental conditions were targeted in the animal room from the day of arrival until necropsy; however, temporary excursions from these environmental conditions may have occurred on an infrequent basis. All observed ranges were documented in the study file.

Temperature: 20°C with a range of 16°C-22°C
Humidity: 50% with a range of 40-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.) Note: Photoperiod times may have changed due to study-related activities.
Enrichment Enrichment for animals was given from the day of arrival until necropsy. The enrichment included relaxing music, an elevated resting platform, and a variety of stainless steel objects attached inside the cage. Rabbits were also given a Certified Timothy Hay Cube (Bio Serv, Flemington, NJ) each day to aid in the digestive process.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Animals were administered biphenyl in the diet seven days/week on GD 7-28. Dietary administration was used to assess the potential toxicity of biphenyl as oral exposure is the preferred route under relevant guidelines. Compared to gavage administration, advantages of the dietary route of oral exposure include the provision of a stable and consistent systemic exposure (Hannas et al., 2016), reductions in stress, avoidance of potential vehicle-induced confounding effects, and the elimination of potential dosing-related injuries to the animals. Thus, oral dietary administration of the test material in feed represented an appropriate means of exposure and was consistent with other testing in the biphenyl dataset, including the previous rabbit studies.

Dose levels for this study (0, 1500, 3000 and 8000 ppm, which corresponds to 0, 65, 130 and 350 mg/kg/day) were based on results of the probe study and were chosen to provide dose-response data for any potential toxicity observed. The chosen high dose level of 8000 ppm (targeting 350 mg/kg/day) was expected to cause a decrease in feed consumption due to reduced palatability of biphenyl-containing diets and decrease body weight gain, but not cause death or obvious suffering. The lower dose levels were selected to provide dose response data for any toxicity that may be observed among the high-dose group rabbits and to establish a no-observable-adverse-effect level (NOAEL).

The vehicle (Teklad Certified Global 2030C Rabbit Diet (Envigo, Madison, Wisconsin) with 0.5% apple flavoring (Flavoring Systems International, Inc., Cincinnati, Ohio, catalog #428527)) was mixed with biphenyl at the concentrations listed above and formed into pellets at Envigo, Teklad Diets (Madison, Wisconsin). Apple flavoring is an effective and innocuous means of enhancing the palatability of experimental diets in rabbits. This flavor ingredient is listed as “Generally Recognized as Safe” (G.R.A.S.) by the Flavor and Extracts Manufacturers Association (F.E.M.A.) and has been deemed safe for human consumption; therefore, use of this flavoring did not have any impact on the outcome or interpretation of the study. Specific details of the flavoring, including G.R.A.S. status and formulation process, were documented in the study file. The biphenyl concentrations of the diets were not adjusted for purity.

Dose confirmation analyses of all dose levels, plus control, were determined pre-exposure. The homogeneity of the low-dose and the high-dose test diets was determined concurrent with dose confirmation. The method used for analyzing the test material in the diet was gas chromatography with positive electron ionization mass spectrometry detection operating in the selected ion monitoring mode (GC/MS-SIM).

A method validation and stability study has shown biphenyl to be stable for at least 35 days in the feed at dose levels ranging from 0.01 to 1.6% (100 to 16000 ppm). The established concentration range and duration spanned those used in this study. Therefore, additional stability analyses were not conducted.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose confirmation analyses of all dose levels, plus control, were determined pre-exposure. The homogeneity of the low-dose and the high-dose test diets was determined concurrent with dose confirmation. The method used for analyzing the test material in the diet was gas chromatography with positive electron ionization mass spectrometry detection operating in the selected ion monitoring mode (GC/MS-SIM).

A method validation and stability study has shown biphenyl to be stable for at least 35 days in the feed at dose levels ranging from 0.01 to 1.6% (100 to 16000 ppm). The established concentration range and duration spanned those used in this study. Therefore, additional stability analyses were not conducted.
Details on mating procedure:
Each sexually mature virgin female was naturally mated with one buck of the same strain at CRP. The observed day of breeding was considered GD 0. GD 0 body weights and records of mating pairs were provided by the supplier and maintained in the study record. Rabbits arrived in the laboratory on GD 1 or 2.
Duration of treatment / exposure:
Rabbits were treated from GD 7 to 28
Frequency of treatment:
Daily
Duration of test:
28 days
Dose / conc.:
0 ppm
Dose / conc.:
1 500 ppm
Dose / conc.:
3 000 ppm
Dose / conc.:
8 000 ppm
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
The purpose of this study was to evaluate the potential maternal and developmental toxicity of biphenyl in New Zealand White (NZW) rabbits following repeated dietary administration.
Groups of 24-mated female NZW rabbits were administered biphenyl in diet at concentrations of 0, 1500, 3000, or 8000 ppm (targeting 0, 65, 130, or 350 mg/kg/day, respectively) on gestation day (GD) 7 through 28. These concentrations corresponded to time weighted average doses of 0, 62.5, 124, or 301 mg/kg/day. In-life parameters evaluated for all groups included: clinical observations, body weight, body weight gain, and feed consumption. On GD 28 all surviving rabbits were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed, and examined for external and visceral alterations. Also, the heads were examined for craniofacial alterations by serial sectioning in approximately one half of the fetuses in each litter, while skeletal examinations were performed on all fetuses.
Maternal examinations:
Daily Observations
Upon arrival, a cage-side examination was conducted twice daily, approximately at the same time each day. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could have been observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Any animals found dead were necropsied as soon as practical.

Clinical Observations
Upon arrival, clinical observations were conducted on all animals at least once daily at approximately the same time each day. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Body Weights/Body Weight Gains
Body weights were recorded on GD 0 (by the supplier), daily during the dosing period, and on GD 28 (terminal). Statistical analyses of body weights were performed using data collected on GD 0, 7, 10, 13, 16, 20, 24, and 28. Statistical analysis of body weight gains was conducted for the following intervals: GD 0-7, 7-10, 10-13, 13-16, 16-20, 20-24, 24-28, 7-28, and 0-28.

Feed Consumption
Daily feed consumption was recorded and statistically analyzed for all animals from GD 4-28.

Test material intake (TMI expressed as mg/kg/day) was calculated upon completion of the study using test material concentrations in the feed, body weights, and feed consumption data.

Necropsy
On GD 28, all surviving animals (not fasted) were euthanized via intravenous injection of Beuthanasia-D (Schering Corporation, Kenilworth, New Jersey), and a limited gross pathologic examination (necropsy) was performed. The sequence of the maternal necropsies was counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification.
The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. The stomach, liver (with gallbladder) and kidneys were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded, and the weight of the liver (with incised gallbladder), kidneys and gravid uterus were recorded. The ratios of liver and kidney weights to terminal body weight were calculated. Representative sections of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of tissues was not conducted. Transponders were removed and placed in bags with the tissues.
Ovaries and uterine content:
A detailed examination of the reproductive tract was performed and the number and position of implantations, viable fetuses, dead fetuses and resorptions were recorded. Resorptions were classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a “dead fetus” indicated a very recent death as evidenced by a lack of external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea was counted. The uteri of females lacking visible implantations were stained with a 10% aqueous solution of sodium sulfide and examined for evidence of early resorptions in order to verify pregnancy status.
Fetal examinations:
All fetuses were weighed, and given an external examination on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were then euthanized by sublingual oral administration of sodium pentobarbital solution. All fetuses were also given a visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations. The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. The fetuses were sexed by examination of the gonads. Approximately one half of the fetuses in each litter were randomly selected for craniofacial examination. The heads of these fetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue. All fetuses were then preserved in alcohol, eviscerated and stained with Alizarin Red S in order to visualize ossified bone. After staining, skeletons were cleared and a thorough evaluation of the fetal skeleton was conducted.
All fetal alterations were classified as a variation or malformation. A variation was defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation was defined as a permanent structural change that may adversely affect survival, development or function and/or which occurs at a relatively low incidence in the specific species/strain. The maternal necropsy and fetal examinations were conducted such that investigators were blind to treatment group assignment.
Statistics:
Stastical information is in the "other information" section due to the absurdly low character limit in this text box.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal (#165) in the control group was found dead on GD 26 with observations of vulvar discharge and blood in the cage. At necropsy the animal was found to be pregnant with no visible lesions
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Animals in the 8000 ppm group had a treatment-related decrease in body weight gains during the GD 7-10 (68.2%, statistically identified) and 7-28 (11.7%) treatment period compared to controls. Animals in the 8000 ppm group also had a treatment-related, statistically-identified slight decrease (3.4%) when compared to controls and corrected for gravid uterine weight. There were no treatment-related differences in the body weights or body weight gains in the 1500 or 3000 ppm groups when compared to controls
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals in the 8000 ppm group had treatment-related, decreased feed consumption values up to 17.9% compared to controls during the GD 7-28 treatment period. Feed consumption values of the 8000 ppm group were statistically identified at several intervals. There were no treatment-related differences in the amount of feed consumed in the 1500 and 3000 ppm groups when compared to controls.

Compound intake: Animals were given 0, 1500, 3000, or 8000 ppm biphenyl (targeting 0, 65, 130, or 350 mg/kg/day, respectively) in pelleted rabbit feed, which corresponded to time-weighted average doses of 0, 62.5, 124, or 301 mg/kg/day, respectively.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically-identified differences in any of the measured parameters for any treated groups when compared to their respective controls for liver and kidney.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Although fetal body weights were lower in the 8000 ppm group compared to controls, they were determined to be unrelated to treatment due to the minimal difference (-4.4%, sexes combined) and lack of a statistical significance when compared to control.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or statistically-identified skeletal alternations in any dose group. Incidental findings bearing no relationship to treatment included the malformations forked ribs, missing ribs, fused thoracic rib, hemicentric thoracic centra, misaligned thoracic centra, thoracic hemivertebra, and hemicentric lumbar centra, and the variations delayed ossification (DO) hyoid, crooked hyoid, DO sternebrae, extra site of ossification sternebrae, irregular pattern of ossification sternebrae, fused sternebrae, DO thoracic centra, DO pubis, and DO talus. Given that these observations occurred in the control group, occurred at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or statistically-identified visceral alterations in any dose group. Incidental findings bearing no relationship to treatment included the malformations retroesophageal subclavian artery, persistent truncus arteriosus, ventricular septal wall defect, hypoplastic lung, diaphragmatic hernia, and missing gall bladder, and the variations right-sided esophagus, hemorrhage of the thymus, missing caudal lung lobe, supernumerary hepatic lobule, pale spleen, particulate material in the kidney, retrocaval ureter, and paraovarian cyst. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
8 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects
Abnormalities:
no effects observed
Developmental effects observed:
no

Gestational Dam Feed Consumption (g) for Statistically Identified Intervals

Concentration

(ppm)

Gestation Day

7-8

8-9

11-12

12-13

13-14

14-15

15-16

23-24

0

148.1

148.1

146.1

145.7

144.9

144.3

146.4

122.5

1500

149.1

148.2

144.1

147.4

142.1

145.0

143.1

130.8

3000

147.1

146.9

142.1

139.0

138.2

138.4

144.8

120.2

8000

 132.1$

(-10.8%)

 135.0$

(-8.8%)

130.9*

(-10.4%)

125.5$

(-14.1%)

 119.3$

(-17.7%)

 125.1$

(-13.3%)

 129.1$

(-11.8%)

100.6*

(-17.9%)

Bold values were considered treatment-related effects. *Statistically different from control mean by Dunnett’s test, alpha = 0.05.  $Statistically different from control mean by Wilcoxon's test, alpha=0.05.

Percentages are the percent difference from controls.

=======================================================================================================================================

Incidences of Visceral Malformations

Concentration (ppm)

0

1500

3000

8000

Retroesophageal Subclavian Artery

F

L

0/183

0/23

0/202

0/23

0/202

0/23

1/191

1/22

Persistent Truncus Arteriosus

F

L

1/183A

1/23

0/202

0/23

0/202

0/23

0/191

0/22

Ventricular Septal Wall Defect

F

L

1/183A

1/23

0/202

0/23

0/202

0/23

0/191

0/22

Hypoplastic Lung

F

L

0/183

0/23

0/202

0/23

0/202

0/23

2/191B,C

2/22

Diaphragmatic Hernia

F

L

0/183

0/23

0/202

0/23

0/202

0/23

2/191B,C

2/22

Missing Gall Bladder

F

L

0/183

0/23

1/202

1/23

0/202

0/23

0/191

0/22

F = fetuses; L = litters

A,B,CMalformations denoted with the same superscript were noted in a single fetus.

=====================================================================================================================================

Incidences of Skeletal Malformations

Concentration (ppm)

0

1500

3000

8000

Forked Ribs

F

L

0/183

0/23

0/202

0/23

1/202D

1/23

1/191B

1/22

Missing Ribs

F

L

0/183

0/23

0/202

0/23

1/202D

1/23

0/191

0/22

Fused Thoracic Rib

F

L

0/183

0/23

0/202

0/23

0/202

0/23

1/191E

1/22

Hemicentric Thoracic Centra

F

L

0/183

0/23

0/202

0/23

0/202

0/23

2/191E

2/22

Misaligned Thoracic Centra

F

L

0/183

0/23

0/202

0/23

0/202

0/23

1/191E

1/22

Thoracic Hemivertebrae

F

L

0/183

0/23

0/202

0/23

2/202D

1/23

0/191

0/22

Hemicentric Lumbar Centra

F

L

1/183

1/23

0/202

0/23

0/202

0/23

0/191

0/22

F = fetuses; L = litters

B,D,EMalformations denoted with the same superscript were noted in a single fetus.

Conclusions:
Dams in the 8000 ppm group had a treatment-related decrease in body weight gains during the GD 7-10 (68.2%) and 7-28 (11.7%) treatment period compared to controls with concomittant decreases in feed consumption. Dams in the 8000 ppm group also had a treatment-related slight decrease in body weight (3.4%) when compared to controls and corrected for gravid uterine weight. There were no treatment-related differences in body weights, body weights corrected for gravid uterine weight, body weight gains, or the amount of feed consumed in the 1500 or 3000 ppm groups when compared to controls.
There were no maternal treatment-related gross pathological observations and no treatment-related effects on liver or kidney weights in any dose group when compared to controls.
There were no treatment-related effects on pregnancy rates, resorptions/litters with resorptions, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss, fetal sex ratios, fetal body weights or gravid uterine weights at any dose level. There were no treatment-related differences in the incidence of any fetal alteration in any of the treated groups compared to controls.
Under the conditions of this study and based on treatment-related effects at the high dose level including decreased body weight gain and feed consumption, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 3000 ppm. The no-observed-effect level (NOEL) for developmental toxicity was 8000 ppm, the highest concentration tested.
Executive summary:

The purpose of this study was to evaluate the potential maternal and developmental toxicity of biphenyl in New Zealand White (NZW) rabbits following repeated dietary administration.

Groups of 24-mated female NZW rabbits were administered biphenyl in diet at concentrations of 0, 1500, 3000, or 8000 ppm (targeting 0, 65, 130, or 350 mg/kg/day, respectively) on gestation day (GD) 7 through 28. These concentrations corresponded to time weighted average doses of 0, 62.5, 124, or 301 mg/kg/day. In-life parameters evaluated for all groups included: clinical observations, body weight, body weight gain, and feed consumption. On GD 28 all surviving rabbits were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed, and examined for external and visceral alterations. Also, the heads were examined for craniofacial alterations by serial sectioning in approximately one half of the fetuses in each litter, while skeletal examinations were performed on all fetuses.

Dams in the 8000 ppm group had a treatment-related decrease in body weight gains during the GD 7-10 (68.2%) and 7-28 (11.7%) treatment period compared to controls with concomittant decreases in feed consumption. Dams in the 8000 ppm group also had a treatment-related slight decrease in body weight (3.4%) when compared to controls and corrected for gravid uterine weight.  There were no treatment-related differences in body weights, body weights corrected for gravid uterine weight, body weight gains, or the amount of feed consumed in the 1500 or 3000 ppm groups when compared to controls.

There were no maternal treatment-related gross pathological observations and no treatment-related effects on liver or kidney weights in any dose group when compared to controls.

There were no treatment-related effects on pregnancy rates, resorptions/litters with resorptions, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss, fetal sex ratios, fetal body weights or gravid uterine weights at any dose level. There were notreatment-relateddifferences in the incidence of any fetal alteration in any of the treated groups compared to controls.

Under the conditions of this study and based on treatment-related effects at the high dose level including decreased body weight gain and feed consumption, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 3000 ppm.  The no-observed-effect level (NOEL) for developmental toxicity was 8000 ppm, the highest concentration tested.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
acceptable
Additional information

The purpose of this study was to evaluate the potential maternal and developmental toxicity of biphenyl in New Zealand White (NZW) rabbits following repeated dietary administration.

Groups of 24-mated female NZW rabbits were administered biphenyl in diet at concentrations of 0, 1500, 3000, or 8000 ppm (targeting 0, 65, 130, or 350 mg/kg/day, respectively) on gestation day (GD) 7 through 28. These concentrations corresponded to time weighted average doses of 0, 62.5, 124, or 301 mg/kg/day. In-life parameters evaluated for all groups included: clinical observations, body weight, body weight gain, and feed consumption. On GD 28 all surviving rabbits were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed, and examined for external and visceral alterations. Also, the heads were examined for craniofacial alterations by serial sectioning in approximately one half of the fetuses in each litter, while skeletal examinations were performed on all fetuses.

Dams in the 8000 ppm group had a treatment-related decrease in body weight gains during the GD 7-10 (68.2%) and 7-28 (11.7%) treatment period compared to controls with concomittant decreases in feed consumption. Dams in the 8000 ppm group also had a treatment-related slight decrease in body weight (3.4%) when compared to controls and corrected for gravid uterine weight.  There were no treatment-related differences in body weights, body weights corrected for gravid uterine weight, body weight gains, or the amount of feed consumed in the 1500 or 3000 ppm groups when compared to controls.

There were no maternal treatment-related gross pathological observations and no treatment-related effects on liver or kidney weights in any dose group when compared to controls.

There were no treatment-related effects on pregnancy rates, resorptions/litters with resorptions, litter size, numbers ofcorpora luteaor implantations, percent preimplantation loss, percent postimplantation loss, fetal sex ratios, fetal body weights or gravid uterine weights at any dose level. There were notreatment-relateddifferences in the incidence of any fetal alteration in any of the treated groups compared to controls.

Under the conditions of this study and based on treatment-related effects at the high dose level including decreased body weight gain and feed consumption, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 3000 ppm.  The no-observed-effect level (NOEL) for developmental toxicity was 8000 ppm, the highest concentration tested.

Justification for classification or non-classification

Given the above-mentioned information, biphenyl does not need to be classified for toxicity to reproduction or developmental toxicity/teratogenicity.