Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
, F1 offspring was followed to adulthood with continued exposure and assessment of neurologic, immunologic, and reproductive structures and functions. In addition, F0 recovery males and 28-day recovery females was assessed.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): 2,4,6-trimethyl phenol (TMP; CAS No. 527-60-6)
- Lot No. 07/31/01
- Purity: 87.72%
- Physical state: white solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: ca. 63 days old
- Weight at study initiation: 276-300 g for males; 201-225 g for females
- Fasting period before study: not performed
- Housing: individually upon arrival, during the acclimation period , and upon the initiation of the treatment period in solid-bottom polycarbonate cages (8” x 19”x 10.5” high) with stainless-steel wire lids (Laboratory Products, Rochelle Park, NJ) with Sani-Chip® cage litter (P.J. Murphy Forest Products Corp., Montville, NJ. Study animals were housed 2 per cage (1 male: 1 female from the same group) during the mating period. Females were caged individually once they were successfully mated (or at the end of the mating period) and throughout gestation. Females were housed with their litters throughout the lactation period. Randomly selected F1 weanlings, males, and females were singly housed during the postweaning exposure period.
- Diet: Pelleted Purina Certified Rodent Diet® (No. 5002, PMI Feeds, Inc., St. Louis, MO), ad libitum
- Water: Tap water (City of Durham, Department of Water Resources, Durham, NC), ad libitum, in plastics water bottles
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air: air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Mazola®
Details on exposure:
VEHICLE
- Concentration in vehicle: 2, 20 and 40 mg/mL
- Amount of vehicle: 5 mL/kg
Details on mating procedure:
Following the 2-week prebreed exposure, the animals were mated on the basis of 1 male to 1 female, selected randomly within each dose group for a period of 14 days, with no change in mating partners. The observation of vaginal sperm of copulation plug was considered evidence of successful mating. Females were examined daily during the cohabitation period for the presence for sperm or copulation plug in the vaginal tract. The day vaginal sperm (or plug) were observed was designated as gd 0. Once vaginal sperm (or plug) were observed, the male and female from the mating pair were individually housed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses were carried out at the RTI International, Center for Life Science and Toxicology, 3040 Cornwallis Road, Research Traingle Park, NC 27709-2194. No problems were experienced in the preparations of the formulation of test chemical in corn oil at 2 and 40 mg/mL. The storage stability studies showed that the test chemical to be stable for 35 days when stored in amber bottles, protected from light, either at ambient temperature or in a refrigerator.
Duration of treatment / exposure:
- Experimental group, F0: 2 weeks of prebreed, 2 weeks of mating, and 3 weeks of gestation and lactation until necropsy (at least 4 weeks for males and 10 weeks for females)
- Any female that did not show evidence of successful mating after 14 days of cohabitation received continued treatment until gd 26 or delivery occurred.
- Recovery group and 28 days females: 28 days
- Selected F1 offspring: from pnd 22 (day after weaning) until necropsy (at least 7 weeks) at least 1 female and 1 male from each F1 litter, for a total of 10/sex/group, were selected on a random basis to continue treatment.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 10, 100, 200 mg/kg/day (experimental group)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 200 mg/kg/day (recovery group and 28 days females)
Basis:
actual ingested
No. of animals per sex per dose:
- Experimental group, F0: 10 (which resulted in 8 pregnant females)
- Recovery groupand 28 days females: 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 10-day dose range-finding study. In this study, TMP was dosed by gavage to male and female rats for 10 consecutive days at 0, 100, 300, and 1000 mg/kg/day. Body weight loss over the 10-day period was observed for males at 1000 mg/kg/day and females at 300 and 1000 mg/kg/day. In addition, weight gain in males at 300 mg/kg/day was reduced by 43% compared to the controls. Therefore 300 mg/kg/day was considered too toxic for the OECD 422 study.
- Rationale for selecting satellite groups: were used as recovery animals to evaluate recovery from any possible treatment-related effects identified in the high-dose group
- Post-exposure recovery period in satellite groups: 2 weeks

Examinations

Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Observations for mortality were made twice daily, and the general condition of all animals was checked daily.

- DETAILED CLINICAL OBSERVATIONS: Clinical examinations were conducted and recorded daily throughout the course of the study.

- BODY WEIGHT: The body weights of the F0 male rats were determined and recorded initially and then weekly until termination. Body weights of the 28-day females and recovery males and females were recorded on a weekly basis until termination. The body weights of F0 female rats were recorded in the same manner until conformation of mating. During gestation, F0 females were weighed on gestational days (GD) 0, 7, 14, and 20. Dams producing litters were weighed on post natal day (pnd) 0, 4, 7, 14, and 21. Any female that did not show evidence of successful mating after 14 days of cohabitation was continued on the original weekly weighing schedule.

- FOOD CONSUMPTION: Feed consumption was recorded weekly for all F0 parental animals during the 2-week prebreed exposure period and recorded weekly for the 28-day females. During pregnancy of the F0 females, feed consumption was measured for GD 0-7, 4-7, 7-14, and 14-21 (after pnd 14 this was confounded by the pups). Any female that did not show evidence of successful mating after 14 days of cohabitation continued on the original weekly schedule.

- HAEMATOLOGY: Blood was collected, from the tail vein, from 5 randomly selected F0 females per group on the last day of the prebreed exposure period (sd 13). Blood was collected prior to necropsy (sd 28) from 5 randomly selected F0 males per group and from the 28-day females. The parameters measured included evaluation of haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, RBC indices, and prothrombin time (PT), a measure of blood clotting time/potential (Becton Dickinson Fibrometer).

- CLINICAL CHEMISTRY: Blood was collected, from the tail vein, at necropsy (sd 28) from 5 randomly selected F0 males per group and from the 28-day females. Evaluations in serum included sodium, potassium, chloride, glucose, total cholesterol, blood urea nitrogen (BUN), creatinine, total protein and albumin, and 2 enzymes indicative for hepatocellular effects (alanine aminotransferase and aspartate aminotransferase)

- URINALYSIS: Prior to necropsy, 5 F0 males per group, randomly selected, and the 28-day females were singly housed overnight in metabolism cages. The total amount of time the animal was in the chamber and the amount of urine collected was recorded. The urine was evaluated for appearance and by dipstick analysis for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocytes.

- NEUROBEHAVIOURAL EXAMINATION: A functional observational battery (FOB), including home cage observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations, was performed on F0 males and 28-day females prior to dosing and weekly to termination at study day (sd) 28. A FOB was also performed on F0 females prior to dosing and weekly during prebreed, mating, gestation, and lactation and on recovery animals (male and female) prior to dosing, weekly during dosing, and once during the recovery period.
Litter observations:
PARAMETERS EXAMINED PREWEANING PERIOD: All F1 pups were individually counted, sexed, weighed, and examined grossly at birth (pnd 0), at pnd 4, 7, and 14, and at weaning (pnd 21). Anogenital distance was recorded with the individual pup weight on pnd 0. The presence or absence of retained nipples and areolae on the ventrum was recorded for F1 offspring males at pnd 11-13. All pups were examined for physical abnormalities (external developmental malformations) at birth and throughout the preweaning period. Survival indices were calculated on pnd 0, 4, 7, and 14 and at weaning.

PARAMETERS EXAMINED POSTWEANING PERIOD: F1 postweaning observations and procedures for each retained female included examination for VP (from pnd 22 until acquisition of vaginal opening) and determination of estrous cyclicity and normality, evaluated by vaginal smears taken daily the last 3 weeks of the postwean exposure period prior to the scheduled sacrifice. For each retained male offspring, observations for PPS began at 35 days of age and continued until acquisition of PPS. Body weights were recorded on the day of VP and PPS. All retained F1 weanlings were weighed and feed consumption measured once per week until their scheduled demise. Daily F1 mortality and clinical observations were conducted for the F1 animals during the last 3 weeks of the postwean exposure period. FOBs were performed on 5 randomly selected postwean F1 offspring/sex/group once, midway through the postwean period. Grip strength was determined in the last week of the postwean period for the same 5 animals/sex/group. In addition, blood was collected, and hematology, clinical chemistry (f1 males and females), and urinalysis (F1 males only) conducted on 5 F1 animals/sex/group prior to necropsy.

STANDARDISATION OF LITTERS: On pnd 4, the size of each litter was adjusted to 10 by eliminated extra pups by random selection to yield, as nearly possible, 5 males and 5 females per litter. Pups culled to standardize litters were euthanized and subjected to a complete gross necropsy.

GROSS EXAMINATION OF DEAD PUPS: Any F1 pup that appeared moribund or that died during lactation was necropsied, when possible, to investigate the cause of death and to identify internal visceral developmental malformations.

F1 MALE ANDROLOGY: At the time of sacrifice 1 testis from each F1 adult male was frozen at 20°C for subsequent enumeration of testicular homogenization-resistant spermatid heads for high-dose and control males. If treatment-related changes in the number of testicular homogenization resitant- spermatid heads were observed in the high-dose group, then these evaluations were extended to the mid-and low-dose group animals (from retained frozen testes). In addition, 1 cauda epididymis from each F1 male was immediately removed, weighed, and seminal fluid from the cauda assessed for sperm number, motility, and morphology. If treatment-related andrological changes were observed in the high-dose group, then these evaluations were extended to the mid- and then to the low-dose group animals.
Postmortem examinations (parental animals):
- GROSS PATHOLOGY: All F0 parental animals, 28-day females, recovery males and females were subjected to a complete gross necropsy, with selected organs weighted and retained or discarded as appropriate. The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities an their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin. Uretri of F0 females were examined for the number of nidation (implantation) scars.
- Organs weighed and retained for F0 animals, 28-day females and recovery animals: liver, kidneys, adrenals, thymus, heart, brain, spleen, testes, epididymides, prostate, seminal vesicles, ovaries, uterus with cervix + vagina.
- Organs only retained for F0 animals, 28-day females and recovery animals: spinal cord, thyroid, sciatic nerve, stomach, small/large intestine, trachea/lungs, urinary bladder, bone marrow (femur), lymph nodes, gross lesions.

- HISTOPATHOLOGY: Full histopathology was performed on all retained organs from 5 randomly selected F0 males and females in the control and high-dose groups as well as for the 28-day females.
Postmortem examinations (offspring):
- GROSS PATHOLOGY: The retained F1 adults were subjected to a complete gross necropsy, with selected organs weighted and retained or discarded as appropriate. The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities an their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin. All nonselected F1 weanlings were subjected to a complete gross necropsy. The retained F1 offspring were sacrificed at ca. 70 days of age and subjected to the same assessments as the F0 parents (expect for the nidation scars).
- Organs weighed and retained for F1 adult animals: liver, kidneys, adrenals, thymus, heart, brain, spleen, testes, epididymides, prostate, seminal vesicles, ovaries, uterus with cervix + vagina.
- Organs only retained for F1 adult animals: spinal cord, thyroid, sciatic nerve, stomach, small/large intestine, trachea/lungs, urinary bladder, bone marrow (femur), lymph nodes, gross lesions.
- Organs only weighed F1 weanlings: thymus, brain, spleen.
- Organs only retained F1 weanlings: gross lesions.
- Organs weighed and retained F1 weanlings: testes, epididymis, ovaries, uterus with cervix + vagina.

- HISTOPATHOLOGY: Full histopathology was performed on all retained organs from 5 randomly selected F1 males and females.
Statistics:
See any other information on materials and methods incl. tables
Reproductive indices:
The following indices were determined:
- Female mating index (%) = (number of females sperm positive/ number of females paired) x 100
- Female fertility index (%) = (number of females pregnant / number of females sperm positive) x 100
- Gestation index (%) = (number of females with live litters / number of females pregnant) x 100
- Male mating index (%) = (number of males impregnating females / number of males paired) x 100
- Male fertility index (%) = (number of males siring litters / number of males impregnating females) x 100
- Pregnancy index (%) = (number of pregnant females / number of males impregnating females) x 100
Offspring viability indices:
The following indices were determined:
- Live birth index (%) = (number of live pups at birth / total number of pups born) x 100
- 4-Day survival index (%) = (number of pups surviving 4 days (precull) / total number of live pups at birth) x 100
- 7-Day survival index (%) = (number of pups surviving 7 days / total number of live pups at 4 days (postcull)) x 100
- 14-Day survival index (%) = (number of pups surviving 14 days / total number of live pups at 7 days) x 100
- 21-Day survival index (%) = (number of pups surviving 21 days / total number of live pups at 14 days) x 100
- Lactation index (%) = (number of pups surviving 21 days / total number of lie pups at 4 days (postcull)) x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- F0 males: No parental males died on study. Treatment- related clinical observations of F0 males during this period included rooting postdosing in 5 males at 200 mg/kg/day. No other findings exhibited a treatment- or dose-related pattern of incidence or severity.
- 28-day females: All of the 28-day females and the recovery females (each with 5/group at 0 and 200 mg/kg/day) survived to scheduled necropsy. Treatment-related clinical observations were limited to rooting postdosing in all 5 females at 200 mg/kg/day.
- Recovery females: Treatment-related clinical observations of the recovery females were limited to rooting postdosing in all 5 females at 200 mg/kg/day.
- F0 females: All 10 F0 females/group survived to scheduled sacrifice. Treatment-related clinical observations of the F0 females included rooting postdosing in 2, 3, and 10 females at 10, 100, and 200 mg/kg/day, respectively.
- F0 gestation: Treatment-related clinical observations during gestation included rooting postdosing in 1 female at 10 mg/kg/day and in 8 females at 200 mg/kg/day; and salivating prior to dosing in 1 female at 200 mg/kg/day.
- F0 lactation: Treatment-related maternal clinical observations during lactation included rooting postdosing that was observed in 1, 1, 5, and 6 females at 0, 10, 100, and 200 mg/kg/day, respectively.

BODY WEIGHT (PARENTAL ANIMALS)
- F0 males: No treatment-related effects on F0 male body weights were observed. Significantly higher weights for sd 7, 14, 21, and 28 (but not sd 0) occurred at 10 mg/kg/day, with no effects at 100 or 200 mg/kg/day. F0 male body weight change was significantly increased at 10 mg/kg/day for sd 0-7 and unaffected for all other intervals at this dose and for all intervals at 100 and 200 mg/kg/day.
- Recovery males: High-dose recovery males exhibited no effects on body weights on sd 0, 7, 14, 21, and 27 during the 4-week treatment period or on sd 34 or 41 during the 2-week recovery period. Body weight gains were similarly unaffected in the high-dose recovery males for all intervals during the 28-day dosing period and the 2-week recovery period. The only treatment-related clinical sign during the dosing phase was rooting postdosing in 3 males at 200 mg/kg/day.
- 28-day females: There were no differences between groups for body weights on sd 0, 7, 14, 21, and 27 during the 28-day exposure period. Body weight change during this period (sd 0-27) was also equivalent between groups for all intervals and for the entire 28-day period.
- Recovery females: The body weights of the recovery females during exposure (sd 0 through 27) and in the recovery period (sd 28 through 41) were equivalent between the 2 groups for all time points examined (sd 0, 7, 14, 21, 27, 34, and 41). Bodyweight changes for all intervals were equivalent between the 2 groups except for sd 34-41 when the recovery female mean body weight change at 200 mg/kg/day was significantly higher than the value at 0 mg/kg/day.
- F0 females: There were no significant differences among groups for F0 female body weights or body weight changes during the prebreed period (sd 0-14) or for the few females in the control and low-dose groups (10 mg/kg/day) that were not identified as sperm positive during the mating (sd 0-28) or postmating (sd 14-42) periods.
- F0 gestation: There were no significant differences in the F0 maternal body weights on gd 0, 7, 14, or 20. There were also no differences in body weight changes for any interval in any group.
- F0 lactation: There were no significant differences in F0 maternal lactational body weights at any dose for any time point. There were no significant differences in F0 maternal body weight change for any interval across groups.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- F0 males: No treatment-related effects on feed consumption were observed. Feed consumption values, expressed as g/day, were significantly increased at 10 mg/kg/day for sd 0 to 7, 7 to 14, and 0 to 14, with no effects at 100 or 200 mg/kg/day. There were no significant effects of treatment on F0 male feed consumption expressed as g/kg/day at any dose for any interval during sd 0-14.
- 28-day females: Feed consumption in g/day and g/kg body weight/day was equivalent between the 2 groups for all intervals and the entire 28-day dosing period.
- F0 females: There were no significant effects on F0 female feed consumption, expressed as g/day or g/kg/day, in any group during the 2 weeks of the prebreed period.
- F0 gestation: There were no changes across groups for maternal feed consumption expressed as g/day or g/kg body weight/day for any interval during gestation.
- F0 lactation: F0 maternal lactational feed consumption, expressed as g/day and g/kg/day, was unaffected for all intervals in all groups. As anticipated, maternal feed consumption was increased in all groups from pnd 14-21 due to the pups self-feeding.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no significant effects of exposure to TMP on F0 mating, fertility, gestational, or pregnancy indices in F0 parental males and females during the production of F1 offspring. The precoital interval and gestational length were equivalent across all groups. There were also no changes across groups for the number of total implantation sites per litter and the number of total and live pups per litter at birth. There were also no significant differences across groups for percent postimplantation loss per litter or the number of dead pups at birth.

HAEMATOLOGY / CLINICAL CHEMISTRY (PARENTAL ANIMALS)
- F0 males: No clinical chemistry or hematology parameters exhibited treatment- or dose- related changes. Blood urea nitrogen, creatinine, glucose, total cholesterol, aspartate aminotransterase, alanine aminotransferase, sodium, and chloride were unaffected across groups. Total protein and albumin were significantly reduced at 100 and 200 mg/kg/day. Potassium concentrations were significantly increased at 10, 100, and 200 mg/kg/day. There were no statistically significant or biologically relevant differences across groups for any blood parameters, including absolute or corrected white blood cell count, absolute or nucleated red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, hemoglobin or hemoglobin concentrations, red blood cell distribution width, platelet count or volume, percentages of segmented neutrophils, lymphocytes, monocytes, eosinophils, or prothrombin clotting time.
- 28-day females: There were no differences between groups for any of the blood parameters for fluid or cellular endpoints, white blood cell differential counts, prothrombin time.
- F0 females: There were no treatment-related changes on any hematology measurements following the 2-week prebreed exposure. A significant increase in hematocrit at 200 mg/kg/day was observed, which was not considered treatment-related based on a lack of effects on correlating parameters or similar findings in the males at this dose. Also, mean corpuscular hemoglobin was significantly reduced at 100 mg/kg/day but unaffected at 10 and 200 mg/kg/day.

URINALYSIS (PARENTAL ANIMALS)
- F0 males: The specific gravity and pH of urine were equivalent across groups.
- 28-day females: There were no difference between groups for urinary specific gravity or pH. None of the additional parametes exhibitied treatment- or dose-related changes.

NEUROBEHAVIOUR (PARENTAL ANIMALS)
- Baseline F0 males: There were no significant differences for any parameters included in the FOB, including home-cage observations, handling observations, sensory and neuromuscular observations, or open field observations during quarantine for the males assigned to any dose group.
- F0 males: No parameters evaluated in the FOB exhibited any statistically significant or biologically relevant difference across groups. There were no significant changes in any of the FOB tests. There was also no effect of treatment on motor activity, auditory startle, or grip strength which were performed in week 4 prior to scheduled termination. None of the statistical analyses indicated a treatment- or dose-related effect.
- Recovery males: FOB analysis for the recovery males (5/group at 0 and 200 mg/kg/day) during quarantine indicated no effects on body weight or on any parameters evaluated as part of the FOB assessment. FOB evaluations during weeks 1 through 4 and week 7 of the recovery males indicated no differences in body weights or in any parameters evaluated as part of the FOB assessment between males at 0 and 200 mg/kg/day.
- 28-day females: FOB evaluations performed once per week for weeks 1 through 4 indicated no differences between the 2 groups (0 and 200 mg/kg/day) for body weights or any parameters examined in the FOB. Just prior to scheduled necropsy of 28-day females, there were no effects on auditory startle or motor activity. Hindlimb (but not forelimb) grip strength was significantly reduced at 200 mg/kg/day, which was not considered treatment related due to the small magnitude of the change and lack of effects in the F0 males and females.
- Recovery females: FOB evaluations were performed on the recovery females once per week during the 4- week exposure period and once (week 7) during the 2-week recovery period. For all 5 of these FOB evaluations, there were no differences between groups for mean body weights or for any of the parameters evaluated in the FOB assessment.
- F0 females: The F0 females were evaluated in the FOB once per week for 9 weeks to encompass the 2-week prebreed (weeks 1 and 2), mating and early gestation (weeks 3 through 5), and late gestation and lactation (weeks 6 through 9). For all 9 weeks of evaluation, there were no differences among groups for mean body weights or treatment-related effects on any parameters. For week 1, the only FOB parameters with significant differences among groups were pupil size score (significantly reduced percentage with score of 1 at 200 mg/kg/day) and average pupil size score (significantly increased size score at 200 mg/kg/day). For week 2, there were no parameters that differed across groups. For week 3, the only parameter affected was average tail pinch score (significantly reduced at 200 mg/kg/day). For weeks 4 through 9, there were no parameters that differed among groups.

SACRIFICE BODY WEIGHT AND ORGAN WEIGHTS (PARENTAL ANIMALS)
- F0 males: There were no treatment-related effects on organ weights. Sacrifice body weights and absolute weights of the brain, thymus, heart, liver, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, and seminal vesicles with coagulating glands were all unaffected. Absolute prostate weight was significantly reduced at 100 mg/kg/day but statistically equivalent at 10 and 200 mg/kg/day. Organ weights relative to terminal body weights were unaffected for the thymus, heart, liver, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, and seminal vesicles plus coagulating glands. Relative brain weight was significantly reduced at 10 mg/kg/day and unaffected at 100 and 200 mg/kg/day. Relative prostate weight was significantly reduced at 10 and 100 mg/kg/day but unaffected at200 mg/kg/day. Organ weights relative to brain weights were unaffected for the thymus, heart, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, prostate, and seminal vesicles with coagulating glands. Relative liver weight was significantly increased at 100 mg/kg/day and unaffected at 10 and 200 mg/kg/day.
- Recovery males: No treatment-related effects were observed. Terminal body weights and all organ weights (absolute and relative to terminal body weight and to terminal brain weight) were equivalent between the 2 groups except for paired adrenal glands; absolute weight and weight relative to terminal body weight were significantly reduced relative to the control values. Paired adrenal weight relative to brain weight was equivalent between the 2 groups. Since there was no effect on adrenal gland weight following 28 days of dosing, this difference in the recovery group was considered due to random biological variation.
- 28-day females: At scheduled necropsy of the 28-day females, there were no differences between the 2 groups (0 and 200 mg/kg/day, 5 females/group) for terminal body weights or any organ weights (absolute, relative to terminal body weights, or relative to terminal brain weights).
- Recovery females: At scheduled necropsy of the recovery females at the end of the 2-week recovery period, there were no differences between groups (0 and 200 mg/kg/day, 5/group) for terminal body weights or for the weights of any organs, absolute, relative to terminal body weights, or relative to terminal brain weights.
- F0 females: Beginning during week 7, F0 females were necropsied on schedule, based on the weaning date of their litters, so the number of F0 females per group dropped over time.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- F0 males: Gross findings at F0 male necropsy did not exhibit any treatment- or dose-related indices or severities.
- Recovery males: No gross lesions were observed in the organs examined from the recovery males.
- 28-day females: There were no treatment-related gross findings at necropsy.
- Recovery females: There were no treatment-related gross findings at necropsy.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- F0 males: Of the 5 males each at 0 and 200 mg/kg/day, there were no histopathologic changes related to treatment.
- 28-day females: There were no treatment-related microscopic findings in the females from the 200 mg/kg/day group.

REPRODUCTIVE INDICES
There were no significant effects of exposure to TMP on F0 mating, fertility, gestational, or pregnancy indices in F0 parental males and females during the production of F1 offspring.The precoital interval and gestational length were equivalent across all groups. There were also no changes across groups for the number of total implantation sites per litter and the number of total and live pups per litter at birth. There were also no significant differences across groups for percent postimplantation loss per litter or the number of dead pups at birth.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
- F1 males: All 10 F1 males/group survived to scheduled sacrifice.
- F1 females: All 10 F1 females/group survived to scheduled sacrifice.

CLINICAL SIGNS (OFFSPRING)
- F1 males: Treatment­ related clinical observations included rooting postdosing in 2, 2, 7, and 9 F1 males at 0, 10, 100 and 200 mg/kg/day, respectively. Salivating pre-/postdosing was observed in 4 males only at 100 mg/kg/day.
- F1 females: Treatment-related clinical observations included rooting postdosing in 7 females each at 100 and 200 mg/kg/day and salivation prior to dosing in 1, 4, and 2 females at 10, 100, and 200 mg/kg/day, respectively.

BODY WEIGHT (OFFSPRING)
- F1 males: There were no significant differences among groups for body weights during the postweaning period (pnd 22 to 71). Body weight change values were also unaffected across all groups for all intervals from pnd 22 through 78.
- F1 females: There were no significant differences in body weight on pnd 29 through 78 across all dose groups. On pnd 22, the mean F1 female body weight at 200 mg/kg/day was significantly reduced, with no effects at any later time point at this dose or at any time point for the other groups. Body weight change values were unaffected across all groups at all intervals from pnd 22 through 78. At scheduled sacrifice, at 70 days of age, mean body weights were unaffected across all groups.

FOOD CONSUMPTION (OFFSPRING)
- F1 males: There were no differences in feed consumption, expressed as g/day or g/kg/day, for any postwean interval in any group.
- F1 females: Feed consumption values, expressed as g/day and g/kg/day, were equivalent across all dose groups for the F1 females from pnd 22 to 78, except for feed consumption in g/kg/day for pnd 29-36, which was significantly increased at 200 mg/kg/day, with no effect on feed consumption for this interval when expressed as g/day.

SEXUAL MATURATION (OFFSPRING)
- F1 males: F1 male age at acquisition of PPS (both absolute and adjusted for body weight at acquisition) was unaffected across all dose groups. There were no effects across all 4 groups for percent motile sperm or percent progressively motile sperm, epididymal sperm concentration, testicular homogenization-resistant SHCs, daily sperm production (per testis), efficiency of daily sperm production (per gram testis), or percent abnormal sperm. The % abnormal sperm values at 0 mg/kg/day (1.90±0.25) and 200 mg/kg/day (2.45±0.23) were well within historical control values.
- F1 females: F1 female age at acquisition of VP (absolute and relative to body weight at acquisition) was equivalent across all groups.

HAEMATOLOGY / CLINICAL CHEMISTRY (OFFSPRING)
- F1 males: There were also no effects across groups for any blood parameters (fluid and cellular elements), including white blood cell differential counts and prothrombin clotting time.
- F1 females: Blood urea nitrogen, creatinine, total protein, albumin, total cholesterol, aspartate aminotransferase, alanine aminotransferase, sodium, potassium, and chloride were unaffected by treatment. Blood glucose was significantly elevated at 200 mg/kg/day which was not considered treatment related due to the magnitude of the change, lack of effects on other parameters, and lack of similar effects in F0 females and males. White blood cell count, nucleated red blood cell count, corrected white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular haemoglobin concentration, red blood cell distribution width, platelet count, mean platelet volume, segmented neutrophils, lymphocytes, monocytes, eosinophils, and prothrombin time were also unaffected by treatment.

URINALYSIS (OFFSPRING)
- F1 males: Urine specific gravity and pH were equivalent across all groups. None of the additional urinary parameters exhibited treatment- or dose-related changes.

NEUROBEHAVIOUR (OFFSPRING)
- F1 males: FOB was performed once midway through the postweaning period for 5 F1 males/group. There were no significant differences among groups for body weights, home cage observations, handling observations, sensory and neuromuscular observations, or open field observations. Grip strength was also evaluated in 5 F1 males/group during the postwean period. Forelimb grip strength was unaffected across groups. Hindlimb grip strength was significantly reduced at 10 mg/kg/day and unaffected at 100 and 200 mg/kg/day.
- F1 females: FOB was performed once mid-way through the postweaning period for F1 females. There were no significant differences for body weights, home cage observations, handling observations, sensory and neuromuscular observations, or open field observations across all groups. There were no effects on average forelimb or hindlimb grip strength in F1 females at any dose level during the last week of the postwean holding period. The estrous cycle lengths, monitored the last 3 weeks of the postwean period for the F1 females, were equivalent across all dose groups.

ORGAN WEIGHTS (OFFSPRING)
- F1 males: There were no treatment-related effects observed for organ weights. Absolute paired kidney weights (but not weights relative to body or brain weights) were significantly increased at 100 mg/kg/day and unaffected at 10 and 200 mg/kg/day. Liver weight, relative to brain weight (but not weight relative to terminal body weight), was significantly increased at 200 mg/kg/day.
- F1 females: Absolute weights and weights relative to terminal body and brain weights of the brain, thymus, heart, liver, spleen, paired kidneys, paired adrenals, paired ovaries, and uterus with cervix and vagina were equivalent across all groups.

GROSS PATHOLOGY (OFFSPRING)
- There were 10 F1 males/group and 10 F1 females/group at 0, 10, 100, and 200 mg/kg/day, respectively, evaluated at scheduled necropsy.
- F1 males: There were no treatment-related gross finding at necropsy.
- F1 females: There were no treatment-related gross finding at necropsy.

HISTOPATHOLOGY (OFFSPRING)
- F1 males: There were no treatment-related histopathological findings.
- F1 females: There were no treatment-related histopathological findings.

OTHER FINDINGS (OFFSPRING)
- F1 males: On the day before necropsy, 3 males at 100 mg/kg/day were not dosed on sd 78 (1) and 79 (2) due to insufficient dosing solution remaining.
- F1 females: Six females at 10 mg/kg/day and 10 females at 100 mg/kg/day were not dosed on the day prior to necropsy due to insufficient dosing solutions in these 2 group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In the absence of any parental or offspring toxicity, the F0 male and female (either pregnant or nonpregnant) systemic no observable adverse effect level (NOAEL) was at least 200 mg/kg/day. The NOAEL for F0 reproductive toxicity was at least 200 mg/kg/day. The NOAEL for F1 offspring toxicity was also at least 200 mg/kg/day.
Executive summary:

In a GLP compliant combined repeated dose toxicity study with the reproductive/developmental toxicity screening test, performed according to OECD 422, male and female CD (Sprague-Dawley [SD]) F0 rats were administered 2,4,6-Trimethyl Phenol (TMP, Mesitol) orally by gavage. These animals received 0, 10, 100, and 200 mg/kg/day at a dose volume of 5 ml/kg/day in Mazola® corn oil, 10 animals/sex/dose, for 2 weeks of prebreed exposure (males and females), 2 weeks of mating (males and females), and 3 weeks of gestation and lactation each (F0 females) for F0 parents, and direct dosing of selected F1 offspring from weaning through scheduled sacrifice, at least 7 weeks postweaning. Five additional F0 males per group from the control and 200 mg/kg/day groups were designated as recovery animals and held without dosing for 2 weeks after the F0 male dosing period was completed, to evaluate recovery from any possible treatment-related effects identified in the high-dose group. Five additional females each from the 0 and 200 mg/kg/day groups (designated "28-day females") were not mated and were terminated after 28 days of dosing. Similarly, 5 females each from the 0 and 200 mg/kg/day groups (designated "28-day recovery females") were dosed for 28 days and held without dosing for an additional 2 weeks as for the recovery group of males. The F0 males and the 28-day females were, therefore, tested in a protocol consistent with OECD Guideline No. 407. Body weights, feed consumption and clinical signs were recorded. A function observational battery observation was performed on all initial animals once during quarantine and at least once per week for F0 animals during prebreed, mating (both sexes), gestation, and lactation (F0 females) treatment periods and on 5 F1 females and 5 F1 males once midway during the postwean exposure period. After the 2-week prebreed exposure period, animals were randomly mated within treatment groups for a 2-week mating period to produce the F1 generation, with continuing exposure. All F0 parental animals, nonselected F1 weanlings, and retained F1 adults were necropsied with complete histologic evaluation of the 28-day females and for 5 selected F0 and F1 males and females in the 0 and 200 mg/kg/day groups. On the day of birth (postnatal day [pnd] 0), anogenital distance was measured and body weights recorded for all live F1 pups in all litters. F1 litters were culled on pnd 4 to yield, as nearly as possible, 5 males and 5 females per litter. The culled F1 pups were weighed, euthanized, and necropsied with complete external and visceral examinations. For the remaining F1 pups, survival indices were calculated at least weekly through weaning (pnd 21). At weaning, at least 1 female and 1 male (whenever possible) from each F1 litter were randomly selected for a total of 10/sex/group to continue treatment for 7 more weeks, with dosing for F1 selected pups begun on pnd 22 until all pups were at least 70 days of age. F1 postweaning observations and procedures for each retained F1 female included examination for vaginal patency (VP; from pnd 22 until acquisition of vaginal opening) and determination of estrous cyclicity and normality evaluated by vaginal smears taken daily the last 3 weeks of the postwean exposure period prior to scheduled sacrifice. For each retained F1 male offspring, observations for cleavage of the balanoprepreputial gland (preputial separation; PPS) began at 35 days of age and continued until acquisition of PPS. Andrologic assessments were also performed on the F1 retained males at necropsy. In addition, hematology, clinical biochemistry, and urinalysis (28-day females and males only) assays were performed at necropsy for all 28-day females, for 5 randomly selected parental F0 males and females per dose group, and for 5 F1 adult males and females per dose group. There were no indications of toxicity in the recovery males, 28-day females, or recovery females. There were no treatment­ related effects on reproductive parameters. There was no F0 offspring toxicity during lactation in either sex. Acquisition of puberty in F1 males and females was unaffected, and F1 postweanlings exhibited no systemic toxicity in either sex at any dose. In conclusion, in the absence of any parental or offspring toxicity, the F0 male and female (either pregnant or nonpregnant) systemic no observable adverse effect level (NOAEL) was at least 200 mg/kg/day. The NOAEL for F0 reproductive toxicity was at least 200 mg/kg/day. The NOAEL for F1 offspring toxicity was also at least 200 mg/kg/day.