4,4'-isopropylidenedi-2,6-xylol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Crl:CD®(SD) rats (72 males and 60 females) were received from Charles River Laboratories, Inc., Raleigh, North Carolina, on May 8, 2012. Males and females came from separate rooms at the breeding facility to avoid sibling mating. The animals were approximately 59 days old upon receipt and approximately 72 days old on the first day of dosing. The range of F0 male body weights at the start of the prebreed exposure period was 308 g to 389 g and the body weight range for the F0 females was 222 g to 288 g.

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The actual mean daily temperature ranged from 69.6°F to 72.2°F (20.9°C to 22.3°C) and mean daily relative humidity ranged from 40.1% to 66.1%. Fluorescent lighting provided illumination for a 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod.

Each rat was uniquely identified by an eartag displaying the animal number. Following receipt and until pairing, all F0 parental test animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage board, animals not used for pairing (5 males/group in the control and high dose groups) remained in these cages until euthanasia. The 12 F0 rats/sex were paired for mating in the home cage of the male. Following positive evidence of mating, the males remained hosed in suspended wire-mesh cages until their schedule necropsy and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed O’Cobs®, The Andersons, Cob Products Division, Maumee, OH). The F0 females were housed in these cages until lactation day 22 (schedule necropsy). Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages with nesting material until necropsy on post-cohabitation day or post-mating day 25. On postnatal day (PND) 21 F1 pups were housed together by litter for 1 week, then beginning on PND 28, the F1 pups were eartagged and individually housed in suspended wire-mesh cages until schedule necropsy. Enrichment devices (Nylabones® or Nyla Pucks®) were provided to all animals as appropriate throughout the study.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test substance formulations were prepared at concentrations of 0, 2, 20 and 200 mg/mL for the dose levels of 0, 10, 100 and 1000 mg/kg/day, respectively. Dose formulations were not adjusted for the purity of the test substance. Stability and resuspension homogeneity (following 8 and 15 days of room temperature storage) of the test substance formulations in corn oil at concentrations ranging from 1 to 200 mg/mL were established in a previously conducted study. Test substance formulations for the current study were prepared approximately once every 14 days as single formulations for each dose level, divided into aliquots for daily dispensation, and stored at room temperature. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

The selected route of administration for this study was oral (gavage) because this is the preferred route for reproductive tests for regulatory purposes. Historically, this route has been used extensively for studies of this nature. F0 Male and female rats and selected F1 male and female rats were administered TMBPA orally by gavage at concentrations of 0, 10, 100, or 1000 mg/kg/day at a dose volume of 5 ml/kg/day. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose, with the following exception. In order to avoid overdosing the dam, the gestation day 14 body weights were used to calculate the individual dosage volumes for mated F0 females from gestation day 14 through lactation day 0. All animals were dosed at approximately the same time each day.

The vehicle and test substance formulations were administered to F0 animals orally by gavage, via an appropriately sized flexible, Teflon® shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily. The control and test substance formulations were administered once daily to the selected F1 pups (12/sex/group) orally by gavage via appropriately sized flexible, disposable, plastic feeding tubes (Instech Solomon, Plymouth Meeting, PA) during PND 22-34 and an appropriately sized, flexible, Teflon®-shafted stainless steel ball tipped dosing cannula (Natume, Japan) during PND 35-49.

Details on mating procedure:

The 12 F0 generation rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group after 14 days of treatment. All animals were randomly selected for cohabitation and sibling mating was not possible based on the animal ordering procedure. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages. For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity and concentration determinations were collected from the top, middle, and bottom strata of each test substance dosing formulation and from the middle stratum of the control group dosing formulation prepared during the study. One set of samples from the first, fourth, and seventh formulation preparations was subjected to the appropriate analyses.
All analyses were conducted using a validated high performance liquid chromatography method with ultraviolet absorbance detection at a wavelength of 212 nm.

Quadruplicate formulation samples were collected using a syringe and dosing cannula and placed in polypropylene tubes. Two samples from each quadruplicate set were processed for analysis and the remaining 2 samples (back-up samples) were stored at room temperature until discarded (if not needed). Formulation samples were processed by adding MeOH and mixing with vortex action. The processed samples were allowed to set for the corn oil to settle to the bottom of the tubes. The processed samples were further diluted as necessary with diluent in autosampler vials. The vials were capped, and the diluted samples mixed with vortex action.

Samples from each dose formulations prepared for use on study were analyzed by single injection (10 microL) using high performance liquid chromatography (HPLC) equipped with a variable wavelength detector at a wavelength of 212 nm, autosamples and Dionex Chromeleon® software. The column was a Phenomenex Luna® C18(2) 100 Å 150 x 4.6 mm, 3-microm particle-size. The retention time of the test substance was approximately 8.9 minutes.

Single injections were made of each calibration standard and processed QC and formulation sample. A calibration curve was constructed for each set of analyses. Concentrations were calculated from the results of the regression analysis using Dionex Chromeleon® software. The concentration data were transferred to a Microsoft Excel® spreadsheet, where appropriate summary statistics, i.e., mean, standard deviation (SD), relative standard deviation (RSD), and concentration as a percent of target concentration, were calculated and presented in tabular form. The concentrations of the formulation and QC samples were calculated by applying any necessary factors to correct for dilution and/or unit conversion.

The acceptance criteria for homogeneity, was that the relative standard deviation (RSD) for the mean concentration must be ≤10% at a concentration within the acceptable limits (85% to 115% of the target concentration). The acceptance criteria for concentrations of corn oil formulations, was that the analyzed concentration must be within 85% to 115% of the target concentration.

Duration of treatment / exposure:

F0 Males (main study animals): Two weeks prior to mating, during the two week mating period through 1 day prior to scheduled euthanasia (Study Days 0-27, for a total of 28 doses).
F0 Males (recovery): Study days 0-27 (for a total of 28 doses).
F0 Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39 and 52 doses, respectively.
F0 Females: Two weeks prior to mating, during the two week mating period and through lactation day 21 (the day prior to necropsy) ) for a total of 57 63 doses.
F1 Males and Females: The F1 generation male and female animals were potentially exposed to the test substance in utero, as well as via the milk while nursing. The selected F1 pups (12 sex/group) were directly administered the test substance following weaning (beginning on PND 22) through the day prior to euthanasia (PND 49) for a total of 28 doses.

Frequency of treatment:

once daily

Details on study schedule:

F0 Generation:
The F0 males were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia on study day 28) for a total of 28 doses. Five additional males included in the control and high dose groups to evaluate recovery from any possible treatment-related effects were sacrificed on study day 42 (following a 14-day recovery period after the cessation of dosing on study day 27). The recovery males were not used for breeding nor were they evaluated for reproductive parameters. The F0 females were dosed during study days 0 through the day prior to euthanasia on lactation day 22 (14 days prior to pairing through lactation day 21) for a total of 57 63 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39 and 52 doses, respectively. F0 females with litters were euthanized on lactation day 22. F0 females that did not mate or did not produce a litter were euthanized on post-mating/post-cohabitation day 25. A complete necropsy was performed on all F0 animals, including weighing and retention of selected organs. Microscopic evaluations of selected tissues from selected animals were also performed.

F1 Generation:
All F0 females were allowed to deliver naturally and rear their young to PND 21. On the day parturition was initiated (PND 0), F1 pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date deliver started. On PND 4 litters were culled to 8 pups per litter (4/sex when possible) to reduce variability. Pups were weaned on PND 21. Twelve male and 12 female pups (a minimum of 1 male and 1 female per litter, if possible) were randomly selected prior to weaning to comprise the F1 generation and begin dose administration on PND 22. Non-selected F1 pups were euthanized on PND 21 and a complete necropsy was performed, including selected organ weights. For the selected pups, evaluation for vaginal patency (females) began on PND 25 and balanopreputial separation (male pups) began on PND 35. All surviving F1 males and females were euthanized on PND 50 and a complete necropsy was performed, including weighing and retention of selected organs. Microscopic evaluations of selected tissues from the control and 1000 mg/kg/day animals, and for the one male found dead, were also performed.

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0 parental animals: 12/sex/group; 5 additional males in the control and high concentration (recovery)
F1 selected pups: 12/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were chosen based on a range-finding study, employing doses of 0, 50, 200, and 1000 mg/kg/day, and administered by oral gavage at 5 ml/kg for 14 days.

Positive control:

none

Parental animals: Observations and examinations:

All rats were observed twice daily for moribundity and mortality. Detailed physical examinations were recorded weekly beginning 1 week prior to test substance administration. Each rat was observed for signs of toxicity approximately 1-2 hours following dose administration. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia or other difficulties. Body weights and feed consumption were recorded at least weekly during the prebreed period for both sexes and for females during gestation (gestation days 0, 4, 7, 11, 14, 17, and 20) and lactation (on lactation days 0 [body weights only when possible], 1, 4, 7, 14, 21, and 22 (final body weight).

Functional Observational Battery (FOB) and locomotor activity assessments were recorded for 6 animals/sex/group on study day 25 (males selected for pairing) or on lactation day 21 (females). Evaluations were conducted prior to dose administration. FOB evaluations included observations in the home cage, when handled and in the open field. Additionally, sensory, neuromuscular and physiological observations were recorded. Locomotor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilized a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the amber plastic boxes to decrease the potential for distraction Data were collected in 5-minute epochs and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation.

Oestrous cyclicity (parental animals):

not evaluated

Sperm parameters (parental animals):

At the necropsy of the F0 males selected for pairing the right epididymis was excised and weighed. A sample of sperm obtained from the right cauda epididymis was loaded onto a slide with a 100-µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported. The right epididymis was placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and epididymis from each male were weighed, stored frozen, homogenized, and analyzed for determination of homogenization-resistant spermatid count and calculation of sperm production rate.

Litter observations:

Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring that were found dead from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Pups with external abnormalities that would warrant further skeletal examination were eviscerated and stained for subsequent skeletal evaluation. Findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt, or interfere with normal body function, or may be incompatible with life) as appropriate. A detailed gross necropsy was performed on any pup that was found dead after PND 4 and prior to weaning; tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.

To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. Each pup received a detailed physical examination on PND 1, 4, 7, 14, and 21. Any abnormalities in nursing behavior were recorded. The anogenital distance of all F1 pups was measured on PND 1; the absolute distance and the absolute distance relative to the cube root of body weight were reported for each pup. Pups were individually weighed on PND 1, 4, 7, 14, and 21. Pups were individually sexed on PND 0 (if possible), 1, 4, 7, 14, and 21. Thoracic nipple retention was evaluated for all male pups on PND 11, 12, and 13. Presence of thoracic nipples was considered to be a positive response; absence of thoracic nipples was considered to be a negative response.

Postmortem examinations (parental animals):

A complete necropsy was conducted on all F0 animals, including recovery males, at scheduled termination following euthanasia by carbon dioxide inhalation. The necropsy included examination of the external surface, the external surface of the brain, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Uteri from F0 females with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss and subsequently discarded. The number of former implantation sites was recorded for all F0 females euthanized on lactation day that delivered a litter. Weights were obtained from the following organs of all animals: adrenal glands, brain, epididymides (paired organs weighed separately), heart, kidneys, liver, ovaries (with oviducts), spleen, testes (paired organs weighed separately), thymus gland and thyroids with parathyroids (weighed after fixation). At the time of necropsy a complete list of guideline-required tissues were retained in appropriate fixative for microscopic evaluations. Experimental Pathology Laboratories, Inc. (Sterling, VA) was responsible for processing all tissues. Microscopic examination was performed on all retained tissues from all treatment phase F0 males and females in the control and 1000 mg/kg/day groups. Target tissues of duodenum and/or jejunum were also examined from treatment phase males and females in the 10 and 100 mg/kg/day groups and recovery phase males in the control and 1000 mg/kg/day groups. Furthermore, the duodenum and jejunum from selected treatment phase F0 males in the control and 1000 mg/kg/day groups were examined using Oil red O stains to identify the nature of the vacuolar contents. Microscopic evaluations were also performed on gross lesions from all F0 animals and correlated to macroscopic findings if possible.

Postmortem examinations (offspring):

Intact offspring that were found dead from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Pups with external abnormalities that would warrant further skeletal examination were eviscerated, stained and examined for skeletal abnormalities. A detailed gross necropsy was performed on any pup that was found dead after PND 4 and prior to weaning; tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.
On PND 4, a detailed physical examination of the pups not selected for continuation on study was conducted. These pups were then euthanized by intraperitoneal injection of sodium pentobarbital, and subjected to a gross necropsy; tissues were preserved for possible future histopathological examination only as deemed necessary by the gross findings.
On PND 21, complete necropsies were performed on nonselected F1 pups euthanized by an intraperitoneal injection of sodium pentobarbital. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The following organs were weighed and collected from all animals at necropsy: brain, kidneys, liver, ovaries, spleen, testes with epididymides (paired organ weighed separately), thymus gland and uterus with cervix and vagina (the uterus was weighed with the cervix, the vagina was retained but not weighed). These tissues and all gross lesions from F1 weanlings were preserved in 10% neutral buffered formalin for possible future histopathologic examination; all other tissues and the carcasses were discarded.
On PND 50 all surviving F1 animals were euthanized by carbon dioxide inhalation. All animals found dead and that survived to the scheduled necropsy (PND 50) were subjected to a complete necropsy that included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. Tissues and organs were collected and placed in 10% neutral buffered formalin as described for the F0 animals. The following organs were weighed from all selected F1 animals at scheduled necropsy: adrenal glands, brain, epididymides (paired organ weighed separately), heart, kidneys, liver, ovaries with oviducts, spleen, testes (paired organ weighted separately), thymus gland, thyroid with parathyroids (weighed after fixation). Experimental Pathology Laboratories, Inc. (Sterling, VA) was responsible for processing all tissues. Microscopic examination was performed on all retained tissues from all F1 males and females in the control and 1000 mg/kg/day groups and for the male that was found dead. In addition, evaluation of gross lesions from all groups was performed.

Statistics:

Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor. Parental body weights, body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites and unaccounted-for sites, number of pups born, balanopreputial separation data, vaginal patency data, anogenital distance, presence of areolas/nipples, sperm production rates, epididymal and testicular sperm numbers, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, pre coital intervals, and continuous FOB data values were subjected to a parametric one way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Histopathological findings and FOB parameters that yielded scalar or descriptive data in the test substance-treated groups were compared to the control group using Fisher’s Exact test. Mean litter proportions (percent per litter) of males at birth and postnatal survival, percentages of motile and progressively motile sperm, and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group. BioStat Consultants Inc conducted the analysis of locomotor activity using SAS version 9.2, or higher, software.

Reproductive indices:

The following reproductive indices were calculated: Male Mating Index (%); Male Fertility Index (%); Male copulation Index (%); Female Mating Index (%); Female Fertility Index (%): and Female Conception Index (%).

Offspring viability indices:

The following litter parameters were calculated: Mean live litter size, Postnatal survival between birth and PND 0 or PND 4 (pre-selection) (% per litter); Postnatal survival for all other intervals [PND 0-1, PND 1-4 (pre-selection), PND 4 (post-selection)-7, PND 7-14, PND 14-21 and PND 4 (post-selection)-21] (% per litter).

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

All F0 males and females survived to the scheduled necropsies. Test substance-related clinical findings of clear and red material around the mouth and/or nose were noted at approximately 1-2 hours following dose administration in the 100 and 1000 mg/kg/day group F0 males and/or females generally throughout the treatment period. No other test substance-related clinical findings were noted at the weekly examinations or at approximately 1-2 hours following dose administration.
Test substance-related lower mean body weight gains were noted in the 100 and 1000 mg/kg/day group F0 males compared to the control group throughout the entire treatment period (study days 0-27). As a result, mean body weights in the 100 and 1000 mg/kg/day group males were 4.2% and 5.1% lower, respectively, than the control group on study day 27. Correspondingly lower mean food consumption was noted for males in these groups only during the first week of treatment (study days 0-7). During the recovery period, mean body weights, body weight gains, and food consumption in the1000 mg/kg/day group males were comparable to the control group. Mean body weight losses or lower mean body weight gains and corresponding lower mean food consumption compared to the control group were noted in the 100 and 1000 mg/kg/day group F0 females during the pre-mating period (study days 0-7 and 0-13, respectively). The deficits in mean body weight gain noted in these females were not of sufficient magnitude to affect mean pre-mating body weights. The body weight effect noted in the 100 and 1000 mg/kg/day group F0 females did not persist into the gestation or lactation treatment periods and mean body weight gains and food consumption during the gestation and lactation treatment periods were unaffected by test substance administration. Mean body weights, body weight gains, and food consumption in the 10 mg/kg/day group F0 males and females were unaffected by test substance administration throughout the study.
No test substance related effects were noted during the FOB or locomotor activity evaluations for F0 males and females at any dosage level.
There were no test substance-related alterations in hematology and coagulation, serum chemistry, or urinalysis parameters (males only) in the F0 generation.
No test substance related macroscopic findings were observed at any dosage level at the F0 generation scheduled necropsy. There were no test substance-related effects on organ weights in the F0 males and females.
Test substance-related, minimal to mild vacuolation of the lamina propria in the duodenum and jejunum was noted in the 100 (duodenum only) and 1000 mg/kg/day F0 group males and females at the primary necropsy. These vacuoles were rarely positive with Oil red O stain and suggested lipid accumulation. Persistence of test substance related, minimal vacuolation in the duodenum and jejunum at slightly higher incidence was noted in the 1000 mg/kg/day F0 group males at the recovery necropsy. No correlating clinical pathology alterations or effects on organ weight were noted. Therefore, the toxicological significance of vacuolation in the present study is uncertain. Similar changes were not noted in the F1 generation.
F0 male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test substance administration at all dosage levels. No test substance-related effects were observed on F0 spermatogenesis endpoints in males at any dosage level. Mean numbers of implantation sites, unaccounted-for sites were unaffected by TMBPA dose administration.

Dose descriptor:

other: NOAEL (reproductive toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance related effects were observed on F0 reproductive performance, gestation length, parturition, or spermatogenesis at any dosage level.

Dose descriptor:

other: NOAEL (systemic toxicity)

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

effects observed, treatment-related

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Offspring PND 0 – 21: Mean number of F1 pups born, live litter size, the percentage of F1 males at birth, and F1 postnatal survival in the 10, 100, and 1000 mg/kg/day groups were comparable to the control group values. Mean F1 offspring body weights and body weight gains for the 10, 100, and 1000 mg/kg/day group males and females were unaffected by maternal test substance administration. No test substance related macroscopic findings or effect on organ weights were observed at any dosage level in the F1 pups at the PND 21 necropsies.

Offspring PND 22-50: In the selected F1 generation animals, 1 male in the 10 mg/kg/day group was found dead immediately following blood collection on PND 50; this death was attributed to the anesthesia. All other F1 males and females survived to the scheduled necropsy. In the F1 generation, increased incidences of clear nasal discharge and clear and red material around the mouth were noted in the 1000 mg/kg/day group males and females at approximately 1-2 hours following dose administration generally throughout the treatment period (PND 22-49). No other test substance-related clinical findings were noted at the weekly examinations or at approximately 1-2 hours following dose administration. Mean body weights, body weight gains, and food consumption in all F1 males and females (following weaning) were unaffected by test substance administration throughout the study.
There were no test substance-related alterations in hematology and coagulation, serum chemistry, or urinalysis parameters (males only) in the F1 animals. No test substance related macroscopic findings or effects on organ weights were observed at any dosage level at the scheduled necropsy of the F1 males and females.

F1 developmental parameters: Test substance-related shorter anogenital distance compared to the control group was noted in the 100 and 1000 mg/kg/day group F1 male and female pups on PND 1. A test substance-related presence of thoracic nipples was observed in the 100 and 1000 mg/kg/day group F1 males compared to the absence of thoracic nipples in the control group males. Mean anogenital distance in the 10 mg/kg/day group male and female pups was unaffected by test substance administration. Mean ages and body weights at attainment of balanopreputial separation or vaginal patency for F1 male and females were unaffected by test substance administration at all dosage levels.

Dose descriptor:

other: NOAEL (neonatal toxicity)

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of an effect on postnatal survival and pup body weights

Dose descriptor:

other: NOAEL (systemic toxicity)

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical findings noted in the 1000 mg/kg/day group F1 males and females following test substance administration (PND 22-49)

Reproductive effects observed:

not specified

Body Weight Changes - F0 Males(g)

Dose (mg/kg/day)	0	10	100	1000
Body Weight Changes
Days 13 to 21
Mean	34	28	26*	25**
S.D.	6.6	8.7	7.7	8.7
N	17	12	12	17
Days 0-27 (entire treatment period
Mean	91	88	73*	70**
S.D.	24.2	20.5	10.2	17.1
N	17	12	12	17
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

Body Weight Changes - F0 Recovery Males(g)

Dose (mg/kg/day)	0	10	100	1000
Body Weight Changes
Days 28-41 (recovery period)
Mean	30	NA	NA	33
S.D.	7.8			8.0
N	5			5
None significantly different from control group.

Body Weight/Body Weight Changes – F0 Females (g)

Dose (mg/kg/day)	0	10	100	1000
Body Weight Changes (prebreed period)
Days 0-13
Mean	5	10	-2	-4*
S.D.	5.0	11.8	10.0	7.8
N	12	12	12	12
Body Weights (Gestation)
GD 7
Mean	290	288	273*	283
S.D.	16.2	15.3	10.4	19.5
N	11	10	12	11
GD 20
Mean	403	400	376**	389
S.D.	21.9	16.5	20.2	23.7
N	11	10	12	11
Body Weight Change (Gestation)
Days 0-20
Mean	140	136	127	134
S.D.	13.7	12.8	20.3	17.0
N	11	10	12	11
Body Weights (Lactation)
Lactation day 1
Mean	306	308	283**	287*
S.D.	16.3	17.9	12.4	21.0
N	11	10	12	11
Body Weight Change (Lactation)
Days 1-21
Mean	34	34	52*	64**
S.D.	17.9	14.6	13.3	10.5
N	11	10	12	11
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

Summary of Microscopic Finding – F0 Males

Dose (mg/kg/day)	0	10	100	1000
Duodenum
Total number examined	12	12	12	12
Examined & unremarkable number examined	12	12	7	7
– Vacuolation	0	0	5*	5*
Minimal	NA	NA	5	5
Jejunum
Total number examined	12	0	12	12
Examined & unremarkable number examined	12	0	12	11
– Vacuolation	0	0	0	1
Mild	NA	NA	NA	1
* Significantly different from control group at the 0.05 level using 2-tailed Fisher’s Exact Test.

Summary of Microscopic Finding – F0 Recovery Males

Dose (mg/kg/day)	0	10	100	1000
Duodenum
Total number examined	5	NA	NA	5
Examined & unremarkable number examined	5			1
– Vacuolation	0			4*
Minimal	NA			4
Jejunum
Total number examined	5	NA	NA	5
Examined & unremarkable number examined	5			2
– Vacuolation	0			3
Minimal	NA			3
NA = not applicable * Significantly different from control group at the 0.05 level using 2-tailed Fisher’s Exact Test..

Summary of Microscopic Finding – F0 Females – Lactation Day 22

Dose (mg/kg/day)	0	10	100	1000
Duodenum
Total number examined	11	10	12	11
Examined & unremarkable number examined	11	10	10	6
– Vacuolation	0	0	2	5*
Minimal	NA	NA	2	5
Jejunum
Total number examined	11	10	12	11
Examined & unremarkable number examined	11	10	12	8
– Vacuolation	0	0	0	3
Minimal	NA	NA	NA	3
* Significantly different from control group at the 0.05 level using 2-tailed Fisher’s Exact test. NA = not applicable

Anogenital Distance (MM) – F1 Rats

Dose (mg/kg/day)	0	10	100	1000
Males
Day 1
Mean	4.30	4.26	4.05*	3.92**
S.D.	0.187	0.138	0.264	0.211
N	11	10	12	11
Females
Day 1
Mean	2.24	2.21	2.06**	2.08*
S.D.	0.093	0.085	0.151	0.140
N	11	10	12	11
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

 

Anogenital Distance Relative to Cube Root of Pup Body Weight – F1 Rats

Dose (mg/kg/day)	0	10	100	1000
Males
Day 1
Mean	2.26	2.26	2.12**	2.09**
S.D.	0.117	0.090	0.125	0.111
N	11	10	12	11
Females
Day 1
Mean	1.19	1.19	1.10**	1.14
S.D.	0.060	0.060	0.083	0.088
N	11	10	12	11
** Significantly different from the control group at 0.01 using Dunnett’s test

 

Areolas/Nipple Retention– F1 Males

Dose (mg/kg/day)	0	10	100	1000
Day 11 (% Positive)
Mean	0.0	2.5	7.0	2.3
S.D.	0.00	7.91	19.42	7.54
N	11	10	12	11
Day 12 (% Positive)
Mean	0.0	0.0	11.1	21.2*
S.D.	0.00	0.00	29.59	35.23
N	11	10	12	11
Day 13 (% Positive)
Mean	0.0	0.0	2.8	20.5*
S.D.	0.00	0.00	9.61	35.03
N	11	10	12	11
** Significantly different from the control group at 0.01 using Dunnett’s test

 

 

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Additional information

In a GLP/ OECD Guideline 422 study with TMBPA no F0 animals died during the study. Clinical findings, mean body weight losses or lower mean body weight gains, and lower mean food consumption were noted in the 100 and 1000 mg/kg/day group F0 males and females. In addition, minimal to mild vacuolation of the lamina propria in the duodenum and jejunum was noted in the 100 (duodenum only) and 1000 mg/kg/day group F0 males and females at the primary necropsy and the persistence of minimal vacuolation in the duodenum and jejunum at slightly higher incidence was noted in the 1000 mg/kg/day group F0males at the recovery necropsy. Therefore, the NOAEL for F0 systemic toxicity is 10 mg/kg/d.

No test substance-related effects were observed on F0 male and female reproductive performance (i.e. mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus), gestation length, parturition, or spermatogenesis at any dosage level. Therefore, under the conditions of this test the NOAEL for reproductive toxicity was 1000 mg/kg/d. Based on the results of this study, TMBPA is not considered a reproductive toxicant.

Short description of key information:
In a GLP/ OECD Guideline 422 study (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), TMBPA was administered by gavage once daily at 0, 10, 100, or 1000 mg/kg bw/d to parental F0 CD® (SD) rats, 12/sex/group, through pre-breed (males & females), mating (males & females), gestation (females only), and lactation to post-natal day (PND) 21 (females only). In addition, 5 additional males per group included in the 0 and 1000 mg/kg bw/d dose groups were designated as recovery animals and held without dosing for two weeks after the dosing period. Selected F1 pups (12/sex/group) were administered TMBPA via gavage at the same dosages as the F0 generation from PND 22 through PND 49 (one day prior to sacrifice on PND 50).

No F0 animals died during the study. Adult F0 toxicity was observed at 100 and 1000 mg/kg/d based on clinical findings, mean body weight losses or significant reductions in body weight gains and lower mean food consumption. Therefore, the NOAEL for F0 systemic toxicity is 10 mg/kg/d.
No test substance-related effects were observed on F0 male and female reproductive performance, gestation length, parturition, or spermatogenesis at any dosage level. Therefore, the NOAEL for reproductive toxicity was 1000 mg/kg/d. Based on the absence of an effect on postnatal survival and pup body weights, the NOAEL for F1 neonatal toxicity was considered to be 1000 mg/kg/day. Based on the shorter anogenital distances noted in the 100 and 1000 mg/kg/day group F1 male pups, and the presence of thoracic nipples in the 100 and 1000 mg/kg/day F1 males, the NOAEL for F1 developmental toxicity was considered to be 10 mg/kg/day. Clinical findings were noted in the 1000 mg/kg/day group F1 males and females following test substance administration (PND 22-49); therefore, the NOAEL for F1 systemic toxicity was considered to be 100 mg/kg/day.

Effects on developmental toxicity

Description of key information

In a GLP/ OECD Guideline 422 study (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test), TMBPA was administered by gavage once daily at 0, 10, 100, or 1000 mg/kg bw/d to parental F0 CD® (SD) rats, 12/sex/group, through pre-breed (males & females), mating (males & females), gestation (females only), and lactation to post-natal day (PND) 21 (females only).  In addition, 5 additional males per group included in the 0 and 1000 mg/kg bw/d dose groups were designated as recovery animals and held without dosing for two weeks after the dosing period.  Selected F1 pups (12/sex/group) were administered TMBPA via gavage at the same dosages as the F0 generation from PND 22 through PND 49 (one day prior to sacrifice on PND 50).
No F0 animals died during the study.  Adult F0 toxicity was observed at 100 and 1000 mg/kg/d based on clinical findings, mean body weight losses or significant reductions in body weight gains and lower mean food consumption.  Therefore, the NOAEL for F0 systemic toxicity is 10 mg/kg/d.  
No test substance-related effects were observed on F0 male and female reproductive performance, gestation length, parturition, or spermatogenesis at any dosage level.  Therefore, the NOAEL for reproductive toxicity was 1000 mg/kg/d.  Based on the absence of an effect on postnatal survival and pup body weights, the NOAEL for F1 neonatal toxicity was considered to be 1000 mg/kg/day.  Based on the shorter anogenital distances noted in the 100 and 1000 mg/kg/day group F1 male pups, and the presence of thoracic nipples in the 100 and 1000 mg/kg/day F1 males, the NOAEL for F1 developmental toxicity was considered to be 10 mg/kg/day.  Clinical findings were noted in the 1000 mg/kg/day group F1 males and females following test substance administration (PND 22-49); therefore, the NOAEL for F1 systemic toxicity was considered to be 100 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)

Details on test animals or test system and environmental conditions:

Crl:CD®(SD) rats (72 males and 60 females) were received from Charles River Laboratories, Inc., Raleigh, North Carolina, on May 8, 2012. Males and females came from separate rooms at the breeding facility to avoid sibling mating. The animals were approximately 59 days old upon receipt and approximately 72 days old on the first day of dosing. The range of F0 male body weights at the start of the prebreed exposure period was 308 g to 389 g and the body weight range for the F0 females was 222 g to 288 g.

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The actual mean daily temperature ranged from 69.6°F to 72.2°F (20.9°C to 22.3°C) and mean daily relative humidity ranged from 40.1% to 66.1%. Fluorescent lighting provided illumination for a 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod.

Each rat was uniquely identified by an eartag displaying the animal number. Following receipt and until pairing, all F0 parental test animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage board, animals not used for pairing (5 males/group in the control and high dose groups) remained in these cages until euthanasia. The 12 F0 rats/sex were paired for mating in the home cage of the male. Following positive evidence of mating, the males remained hosed in suspended wire-mesh cages until their schedule necropsy and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed O’Cobs®, The Andersons, Cob Products Division, Maumee, OH). The F0 females were housed in these cages until lactation day 22 (schedule necropsy). Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages with nesting material until necropsy on post-cohabitation day or post-mating day 25. On postnatal day (PND) 21 F1 pups were housed together by litter for 1 week, then beginning on PND 28, the F1 pups were eartagged and individually housed in suspended wire-mesh cages until schedule necropsy. Enrichment devices (Nylabones® or Nyla Pucks®) were provided to all animals as appropriate throughout the study.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test substance formulations were prepared at concentrations of 0, 2, 20 and 200 mg/mL for the dose levels of 0, 10, 100 and 1000 mg/kg/day, respectively. Dose formulations were not adjusted for the purity of the test substance. Stability and resuspension homogeneity (following 8 and 15 days of room temperature storage) of the test substance formulations in corn oil at concentrations ranging from 1 to 200 mg/mL were established in a previously conducted study. Test substance formulations for the current study were prepared approximately once every 14 days as single formulations for each dose level, divided into aliquots for daily dispensation, and stored at room temperature. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The first dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

The selected route of administration for this study was oral (gavage) because this is the preferred route for reproductive tests for regulatory purposes. Historically, this route has been used extensively for studies of this nature. F0 Male and female rats were administered TMBPA orally by gavage at concentrations of 0, 10, 100, or 1000 mg/kg/day at a dose volume of 5 ml/kg/day. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose, with the following exception. In order to avoid overdosing the dam, the gestation day 14 body weights were used to calculate the individual dosage volumes for mated F0 females from gestation day 14 through lactation day 0. All animals were dosed at approximately the same time each day.

The vehicle and test substance formulations were administered to F0 animals orally by gavage, via an appropriately sized flexible, Teflon® shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for homogeneity and concentration determinations were collected from the top, middle, and bottom strata of each test substance dosing formulation and from the middle stratum of the control group dosing formulation prepared during the study. One set of samples from the first, fourth, and seventh formulation preparations was subjected to the appropriate analyses.
All analyses were conducted using a validated high performance liquid chromatography method with ultraviolet absorbance detection at a wavelength of 212 nm.

Quadruplicate formulation samples were collected using a syringe and dosing cannula and placed in polypropylene tubes. Two samples from each quadruplicate set were processed for analysis and the remaining 2 samples (back-up samples) were stored at room temperature until discarded (if not needed). Formulation samples were processed by adding MeOH and mixing with vortex action. The processed samples were allowed to set for the corn oil to settle to the bottom of the tubes. The processed samples were further diluted as necessary with diluent in autosampler vials. The vials were capped, and the diluted samples mixed with vortex action.

Samples from each dose formulations prepared for use on study were analyzed by single injection (10 microL) using high performance liquid chromatography (HPLC) equipped with a variable wavelength detector at a wavelength of 212 nm, autosamples and Dionex Chromeleon® software. The column was a Phenomenex Luna® C18(2) 100 Å 150 x 4.6 mm, 3-microm particle-size. The retention time of the test substance was approximately 8.9 minutes.

Single injections were made of each calibration standard and processed QC and formulation sample. A calibration curve was constructed for each set of analyses. Concentrations were calculated from the results of the regression analysis using Dionex Chromeleon® software. The concentration data were transferred to a Microsoft Excel® spreadsheet, where appropriate summary statistics, i.e., mean, standard deviation (SD), relative standard deviation (RSD), and concentration as a percent of target concentration, were calculated and presented in tabular form. The concentrations of the formulation and QC samples were calculated by applying any necessary factors to correct for dilution and/or unit conversion.

The acceptance criteria for homogeneity, was that the relative standard deviation (RSD) for the mean concentration must be ≤10% at a concentration within the acceptable limits (85% to 115% of the target concentration). The acceptance criteria for concentrations of corn oil formulations, was that the analyzed concentration must be within 85% to 115% of the target concentration.

Details on mating procedure:

All F0 animals were randomly selected for cohabitation and sibling mating was not possible based on the animal ordering procedure. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages. For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

Duration of treatment / exposure:

F0 Males (used for mating): Two weeks prior to mating, during the two week mating period through 1 day prior to scheduled euthanasia (Study Days 0-27, for a total of 28 doses).
F0 Females: Two weeks prior to mating, during the two week mating period and through lactation day 21 (the day prior to necropsy) ) for a total of 57 63 doses.
F0 Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39 and 52 doses, respectively.

F1 Males and Females: The F1 generation male and female animals were potentially exposed to the test substance in utero, as well as via the milk while nursing. Selected F1 pups (12 sex/group) were directly administered the test substance following weaning (beginning on PND 22) through the day prior to euthanasia (PND 49) for a total of 28 doses.

Frequency of treatment:

once daily

Duration of test:

Through lactation day 22 for F0 females.
Through postnatal day 50 for selected F1 pups.

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

F0 females: 12/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were chosen based on a range-finding study, employing doses of 0, 50, 200, and 1000 mg/kg/day, and administered by oral gavage at 5 ml/kg for 14 days.

The F0 males were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia on study day 28) for a total of 28 doses. The F0 females were dosed during study days 0 through the day prior to euthanasia on lactation day 22 (14 days prior to pairing through lactation day 21) for a total of 57 63 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39 and 52 doses, respectively. F0 females with litters were euthanized on lactation day 22. F0 females that did not mate or did not produce a litter were euthanized on post-mating/post-cohabitation day 25. A complete necropsy was performed on all F0 animals, including weighing and retention of selected organs. Microscopic evaluations of selected tissues from selected animals were also performed.

On the day parturition was initiated (PND 0), F1 pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date deliver started. On PND 4 litters were culled to 8 pups per litter (4/sex when possible) to reduce variability. Pups were weaned on PND 21. Twelve male and 12 female pups (a minimum of 1 male and 1 female per litter, if possible) were randomly selected prior to weaning to comprise the F1 generation and begin dose administration on PND 22. Non-selected F1 pups were euthanized on PND 21 and a complete necropsy was performed, including selected organ weights. For the selected pups, evaluation for vaginal patency (females) began on PND 25 and balanopreputial separation (male pups) began on PND 35. All surviving F1 males and females were euthanized on PND 50 and a complete necropsy was performed, including weighing and retention of selected organs. Microscopic evaluations of selected tissues from the control and 1000 mg/kg/day animals, and for the one male found dead, were also performed.

Maternal examinations:

All rats were observed twice daily for moribundity and mortality. Detailed physical examinations were recorded weekly beginning 1 week prior to test substance administration. Each rat was observed for signs of toxicity approximately 1-2 hours following dose administration. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia or other difficulties. Body weights and feed consumption were recorded at least weekly during the prebreed period for both sexes and for females during gestation (gestation days 0, 4, 7, 11, 14, 17, and 20) and lactation (on lactation days 0 [body weights only when possible], 1, 4, 7, 14, 21, and 22 (final body weight).

Functional Observational Battery (FOB) and locomotor activity assessments were recorded for 6 animals/sex/group on lactation day 21 (females). Evaluations were conducted prior to dose administration. FOB evaluations included observations in the home cage, when handled and in the open field. Additionally, sensory, neuromuscular and physiological observations were recorded. Locomotor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilized a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the amber plastic boxes to decrease the potential for distraction Data were collected in 5-minute epochs and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation.

Ovaries and uterine content:

A complete necropsy was conducted on all F0 females on lactation day 22, at scheduled termination following euthanasia by carbon dioxide inhalation. The necropsy included examination of the external surface, the external surface of the brain, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Uteri from F0 females with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss and subsequently discarded. The number of former implantation sites was recorded for all F0 females euthanized on lactation day that delivered a litter.

Additional F0 female postmortem procedures: Weights were obtained from the following organs of all animals: adrenal glands, brain, epididymides (paired organs weighed separately), heart, kidneys, liver, ovaries (with oviducts), spleen, testes (paired organs weighed separately), thymus gland and thyroids with parathyroids (weighed after fixation). At the time of necropsy a complete list of guideline-required tissues were retained in appropriate fixative for microscopic evaluations. Experimental Pathology Laboratories, Inc. (Sterling, VA) was responsible for processing all tissues. Microscopic examination was performed on all retained tissues from all treatment phase F0 males and females in the control and 1000 mg/kg/day groups. Target tissues of duodenum and/or jejunum were also examined from treatment phase males and females in the 10 and 100 mg/kg/day groups. Furthermore, the duodenum and jejunum from selected treatment phase F0 males in the control and 1000 mg/kg/day groups were examined using Oil red O stains to identify the nature of the vacuolar contents. Microscopic evaluations were also performed on gross lesions from all F0 animals and correlated to macroscopic findings if possible.

Fetal examinations:

No fetal examinations were conducted as each dam was allowed to deliver naturally. On the day parturition was initiated (PND 0), F1 pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date deliver started. Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring that were found dead from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. There were no pups with external abnormalities that warranted further skeletal examination. Findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt, or interfere with normal body function, or may be incompatible with life) as appropriate.

To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. Each pup received a detailed physical examination on PND 1, 4, 7, 14, and 21. Any abnormalities in nursing behavior were recorded. The anogenital distance of all F1 pups was measured on PND 1; the absolute distance and the absolute distance relative to the cube root of body weight were reported for each pup. Pups were individually weighed on PND 1, 4, 7, 14, and 21. Pups were individually sexed on PND 0 (if possible), 1, 4, 7, 14, and 21. Thoracic nipple retention was evaluated for all male pups on PND 11, 12, and 13. Presence of thoracic nipples was considered to be a positive response; absence of thoracic nipples was considered to be a negative response.

On PND 4, a detailed physical examination of the pups not selected for continuation on study was conducted. These pups were then euthanized by intraperitoneal injection of sodium pentobarbital, and subjected to a gross necropsy; tissues were preserved for possible future histopathological examination only as deemed necessary by the gross findings. A detailed gross necropsy was performed on any pup that was found dead after PND 4 and prior to weaning; tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.

On PND 21, complete necropsies were performed on nonselected F1 pups euthanized by an intraperitoneal injection of sodium pentobarbital. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The following organs were weighed and collected from all animals at necropsy: brain, kidneys, liver, ovaries, spleen, testes with epididymides (paired organ weighed separately), thymus gland and uterus with cervix and vagina (the uterus was weighed with the cervix, the vagina was retained but not weighed). These tissues and all gross lesions from F1 weanlings were preserved in 10% neutral buffered formalin for possible future histopathologic examination; all other tissues and the carcasses were discarded.

Statistics:

Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor. Parental body weights, body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites and unaccounted-for sites, number of pups born, balanopreputial separation data, vaginal patency data, anogenital distance, presence of areolas/nipples, sperm production rates, epididymal and testicular sperm numbers, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, pre coital intervals, and continuous FOB data values were subjected to a parametric one way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Histopathological findings and FOB parameters that yielded scalar or descriptive data in the test substance-treated groups were compared to the control group using Fisher’s Exact test. Mean litter proportions (percent per litter) of males at birth and postnatal survival, percentages of motile and progressively motile sperm, and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group. BioStat Consultants Inc conducted the analysis of locomotor activity using SAS version 9.2, or higher, software.

Indices:

Reproductive indices: The following reproductive indices were calculated: Male Mating Index (%); Male Fertility Index (%); Male copulation Index (%); Female Mating Index (%); Female Fertility Index (%): and Female Conception Index (%).

Offspring viability indices: The following litter parameters were calculated: Mean live litter size, Postnatal survival between birth and PND 0 or PND 4 (pre-selection) (% per litter); Postnatal survival for all other intervals [PND 0-1, PND 1-4 (pre-selection), PND 4 (post-selection)-7, PND 7-14, PND 14-21 and PND 4 (post-selection)-21] (% per litter).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
All F0 males and females survived to the scheduled necropsies. Test substance-related clinical findings of clear and red material around the mouth and/or nose were noted at approximately 1-2 hours following dose administration in the 100 and 1000 mg/kg/day group F0 males and/or females generally throughout the treatment period. No other test substance-related clinical findings were noted at the weekly examinations or at approximately 1-2 hours following dose administration.

Mean body weight losses or lower mean body weight gains and corresponding lower mean food consumption compared to the control group were noted in the 100 and 1000 mg/kg/day group F0 females during the pre-mating period (study days 0-7 and 0-13, respectively). The deficits in mean body weight gain noted in these females were not of sufficient magnitude to affect mean pre-mating body weights. The body weight effect noted in the 100 and 1000 mg/kg/day group F0 females did not persist into the gestation or lactation treatment periods and mean body weight gains and food consumption during the gestation and lactation treatment periods were unaffected by test substance administration. Mean body weights, body weight gains, and food consumption in the 10 mg/kg/day group F0 females were unaffected by test substance administration throughout the study. No test substance related effects were noted during the FOB or locomotor activity evaluations for F0 females at any dosage level.

There were no test substance-related alterations in hematology and coagulation or serum chemistry in the F0 females. At scheduled necropsy, no test substance related macroscopic findings or effects on organ weights were observed at any dosage level

Test substance-related, minimal to mild vacuolation of the lamina propria in the duodenum and jejunum was noted in the 100 (duodenum only) and 1000 mg/kg/day F0 group males and females at the primary necropsy. These vacuoles were rarely positive with Oil red O stain and suggested lipid accumulation. Persistence of test substance related, minimal vacuolation in the duodenum and jejunum at slightly higher incidence was noted in the 1000 mg/kg/day F0 group males at the recovery necropsy. No correlating clinical pathology alterations or effects on organ weight were noted. Therefore, the toxicological significance of vacuolation in the present study is uncertain. Similar changes were not noted in the F1 generation.

F0 male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test substance administration at all dosage levels. No test substance-related effects were observed on F0 spermatogenesis endpoints in males at any dosage level. Mean numbers of implantation sites, unaccounted-for sites were unaffected by TMBPA dose administration.

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Offspring PND 0 – 21: Mean number of F1 pups born, live litter size, the percentage of F1 males at birth, and F1 postnatal survival in the 10, 100, and 1000 mg/kg/day groups were comparable to the control group values. Mean F1 offspring body weights and body weight gains for the 10, 100, and 1000 mg/kg/day group males and females were unaffected by maternal test substance administration. No test substance related macroscopic findings or effect on organ weights were observed at any dosage level in the F1 pups at the PND 21 necropsies.

F1 developmental parameters: Test substance-related shorter anogenital distance compared to the control group was noted in the 100 and 1000 mg/kg/day group F1 male and female pups on PND 1. A test substance-related presence of thoracic nipples was observed in the 100 and 1000 mg/kg/day group F1 males compared to the absence of thoracic nipples in the control group males. Mean anogenital distance in the 10 mg/kg/day group male and female pups was unaffected by test substance administration. Mean ages and body weights at attainment of balanopreputial separation or vaginal patency for F1 male and females were unaffected by test substance administration at all dosage levels.

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: shorter anogenital distances noted in the 100 and 1000 mg/kg/day group F1 male and female pups, and the presence of thoracic nipples in the 100 and 1000 mg/kg/day F1 males.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: clinical findings noted in the 1000 mg/kg/day group F1 males and females following test substance administration (PND 22-49)

Abnormalities:

not specified

Developmental effects observed:

not specified

Body Weights and Body Weight Changes – F0 Females (g)

Dose (mg/kg/day)	0	10	100	1000
Body Weight Changes (prebreed period)
Days 0-13
Mean	5	10	-2	-4*
S.D.	5.0	11.8	10.0	7.8
N	12	12	12	12
Body Weights (Gestation)
GD 7
Mean	290	288	273*	283
S.D.	16.2	15.3	10.4	19.5
N	11	10	12	11
GD 20
Mean	403	400	376**	389
S.D.	21.9	16.5	20.2	23.7
N	11	10	12	11
Body Weight Change (Gestation)
Days 0-20
Mean	140	136	127	134
S.D.	13.7	12.8	20.3	17.0
N	11	10	12	11
Body Weights (Lactation)
Lactation day 1
Mean	306	308	283**	287*
S.D.	16.3	17.9	12.4	21.0
N	11	10	12	11
Body Weight Change (Lactation)
Days 1-21
Mean	34	34	52*	64**
S.D.	17.9	14.6	13.3	10.5
N	11	10	12	11
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

 

Summary of Microscopic Finding – F0 Females – Lactation Day 22

Dose (mg/kg/day)	0	10	100	1000
Duodenum
Total number examined	11	10	12	11
Examined & unremarkable number examined	11	10	10	6
– Vacuolation	0	0	2	5*
Minimal	NA	NA	2	5
Jejunum
Total number examined	11	10	12	11
Examined & unremarkable number examined	11	10	12	8
– Vacuolation	0	0	0	3
Minimal	NA	NA	NA	3
* Significantly different from control group at the 0.05 level using 2 -tailed Fisher’s Exact test. NA = not applicable

 

Anogenital Distance (MM) – F1 Rats

Dose (mg/kg/day)	0	10	100	1000
Males
Day 1
Mean	4.30	4.26	4.05*	3.92**
S.D.	0.187	0.138	0.264	0.211
N	11	10	12	11
Females
Day 1
Mean	2.24	2.21	2.06**	2.08*
S.D.	0.093	0.085	0.151	0.140
N	11	10	12	11
* Significantly different from the control group at 0.05 using Dunnett’s test ** Significantly different from the control group at 0.01 using Dunnett’s test

 

Anogenital Distance Relative to Cube Root of Pup Body Weight – F1 Rats

Dose (mg/kg/day)	0	10	100	1000
Males
Day 1
Mean	2.26	2.26	2.12**	2.09**
S.D.	0.117	0.090	0.125	0.111
N	11	10	12	11
Females
Day 1
Mean	1.19	1.19	1.10**	1.14
S.D.	0.060	0.060	0.083	0.088
N	11	10	12	11
** Significantly different from the control group at 0.01 using Dunnett’s test

 

  Areolas/Nipple Retention– F1 Males

Dose (mg/kg/day)	0	10	100	1000
Day 11 (% Positive)
Mean	0.0	2.5	7.0	2.3
S.D.	0.00	7.91	19.42	7.54
N	11	10	12	11
Day 12 (% Positive)
Mean	0.0	0.0	11.1	21.2*
S.D.	0.00	0.00	29.59	35.23
N	11	10	12	11
Day 13 (% Positive)
Mean	0.0	0.0	2.8	20.5*
S.D.	0.00	0.00	9.61	35.03
N	11	10	12	11
** Significantly different from the control group at 0.01 using Dunnett’s test

 

Conclusions:

Under the conditions of this screening study of tetramethyl bisphenol A in male and female Crl:CD(SD) rats, no test substance-related effects were observed on F0 reproductive performance, gestation length, parturition, or spermatogenesis at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive toxicity. Clinical findings, mean body weight losses or lower mean body weight gains, and lower mean food consumption were noted in the 100 and 1000 mg/kg/day group F0 males and females. In addition, minimal to mild vacuolation of the lamina propria in the duodenum and jejunum was noted in the 100 (duodenum only) and 1000 mg/kg/day group F0 males and females at the primary necropsy and the persistence of minimal vacuolation in the duodenum and jejunum at slightly higher incidence was noted in the 1000 mg/kg/day group F0males at the recovery necropsy. Therefore, the NOAEL for F0 male and female systemic toxicity was considered to be 10 mg/kg/day. Based on the absence of an effect on postnatal survival and pup body weights, the NOAEL for F1 neonatal toxicity was considered to be 1000 mg/kg/day. Based on the shorter anogenital distances noted in the 100 and 1000 mg/kg/day group F1 male and female pups, and the feminization of the F1 males characterized by the presence of thoracic nipples in the 100 and 1000 mg/kg/day groups, the NOAEL for F1 developmental toxicity was considered to be 10 mg/kg/day. Clinical findings were noted in the 1000 mg/kg/day group F1 males and females following test substance administration (PND 22-49); therefore, the NOAEL for F1 systemic toxicity was considered to be 100 mg/kg/day.

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

10 mg/kg bw/day

Additional information

In a GLP/ OECD Guideline 422 study with TMBPA no F0 animals died during the study. Clinical findings, mean body weight losses or lower mean body weight gains, and lower mean food consumption were noted in the 100 and 1000 mg/kg/day group F0 males and females. In addition, minimal to mild vacuolation of the lamina propria in the duodenum and jejunum was noted in the 100 (duodenum only) and 1000 mg/kg/day group F0 males and females at the primary necropsy and the persistence of minimal vacuolation in the duodenum and jejunum at slightly higher incidence was noted in the 1000 mg/kg/day group F0males at the recovery necropsy. Therefore, the NOAEL for F0 systemic toxicity is 10 mg/kg/d.

Based on the shorter anogenital distances noted in the 100 and 1000 mg/kg/day group F1 male pups, and the presence of thoracic nipples in the 100 and 1000 mg/kg/day F1 males, the NOAEL for F1 developmental toxicity was considered to be 10 mg/kg/day.

Justification for classification or non-classification

No test substance-related effects were observed on F0 male and female reproductive performance, gestation length, parturition, or spermatogenesis at any dosage level. However, shorter anogenital distances and the feminization were noted in the mid and high dose groups F1 male pups. Based on these results, a conclusion regarding classification and labelling cannot be drawn yet. Once additional information is available, classification according to Directive 67/548/EEC and the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 will be added.