4-(α,α-dimethylbenzyl)phenol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-07-12 to 2005-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

the study exceeds the guideline by following the F1 offspring to adulthood and includes assessment of neurologic function.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(α,α-dimethylbenzyl)phenol

EC Number:

209-968-0

EC Name:

4-(α,α-dimethylbenzyl)phenol

Cas Number:

599-64-4

Molecular formula:

C15H16O

IUPAC Name:

4-(α,α-dimethylbenzyl)phenol

Constituent 2

Reference substance name:

290-968-0

IUPAC Name:

290-968-0

Details on test material:

- Name of test material (as cited in study report): p-cumylphenol
- Substance type: active
- Physical state: white crystals
- Analytical purity: 99.9 %
- Storage condition of test material: Ambient (cool, dry)

Test animals

Species:

rat

Strain:

other: CD® (Sprague-Dawley) rats (Crl:CD[SD] IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: P generation: approximately 10 weeks; F1 generation: treatment began on postnatal day 22, the day after weaning.
- Weight at study initiation: (P) Males: 270.9 to 355.4 g; Females: 215.7 to 248.5 g
- Housing: The animals were individually housed 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, with Sani-Chip cage litter. Study animals were housed 2/cage (1 male:1 female from the same dose level) 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. Selected F1 weanlings, males, and females were singly housed during the post weaning exposure period.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%):30 to 70 (3 relative humidity deviations occurred outside this range during the conduct of the study, but they were very brief, minor, rare and did not affect the design, performance or conclusions of the study)
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES:
From: 2002-07-08 (date of arrival), 2002-07-15 (date of first treatment)
To: 2002-11-15

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The purity of the test substance was assumed to be 100 % for dose formulation purposes (i.e., all measurements of the test substance were made by weight and not corrected for purity). Dosing formulations were prepared by suspending the test substance in corn oil. Dose formulations were stirred for at least 30 minutes prior to use.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg/day

Details on mating procedure:

- M/F ratio per cage: Study animals were housed 2/cage (1 male:1 female from the same dose level) during the mating period, with no change in mating partners.
- Length of cohabitation: 14 days
- Proof of pregnancy: The observation of vaginal sperm or copulation plug was considered evidence of successful mating. Females were examined daily during the cohabitation period for the presence of sperm or copulation plug in the vaginal tract. The day vaginal sperm (or plug) were observed was designated as gestation day 0.
- After successful mating each pregnant female was caged: Females were housed individually once they were successfully mated (or at the end of the mating period) and throughout gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to dosing, studies of homogeneity and storage stability under refrigeration and at ambient temperatures of the test substance in corn oil at 1.0 and 60.0 mg/mL were conducted. In addition, stability tests under conditions simulating dosing in the animal room(s) were conducted.
Dose formulations were prepared and concentration verifications were performed monthly. All dose formulations used in the study had analytical values of 95.3 to 110 % of target concentrations. Vehicle control formulations contained no test substance, with a detection limit of 0.03 mg/mL. The storage stability studies showed the test substance to be stable at least for 32 days when stored in amber bottles either at ambient temperature or in a refrigerator. The simulated dosing stability studies showed formulation of the test substance to be stable under conditions that simulated dosing (in corn oil in a beaker open to light and air at ambient temperature) for 4 hours in dose formations prepared at 1.0 and 60.0 mg/g.

Duration of treatment / exposure:

Dosing for the parental animals began on study day 0 and continued until the day before scheduled necropsy [at least 4-week duration for males (2 week pre-breed and 2 week mating period) and 8 to 10 week duration for females (2 week pre-breed through mating, gestation and lactation)]. For the P generation recovery males, dosing began on study day 0 and continued until the end of the dosing period of the P generation males (at least 4 weeks). The recovery males were then held with no dosing for 2 weeks to evaluate recovery. F1 selected pups were treated directly for at least 7 weeks (from weaning (post-natal day 22) to scheduled sacrifice).

Frequency of treatment:

once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks
- Other: Any female that did not show evidence of successful mating after 14 days of cohabitation was weighed weekly, and treatment continued until gd 26 or delivery occurred. If a female without a confirmed gd 0 date was in fact pregnant and delivered a litter, her lactational information was collected. Beginning on gd 20, each female was observed twice daily for evidence of littering. On the day of birth (pnd 0), anogenital distance was measured and body weight recorded for all live F1 pups in all litters. Body weight was recorded for all live pups on pnd 4 prior to culling and euthanasia. The dams were allowed to rear their young to pnd 21. On pnd 21, each litter was weaned. When each F1 litter reached pnd 21, at least 1 male and 1 female pup from each litter was selected, if possible, for a total of 10/sex/group was randomly selected.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 per sex per group were designated as parental animals and 5 additional males in the control and high-dose groups were designated as recovery males.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Target doses were selected based on the results of a 10-day range-finding study.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and once daily for general condition
- Observations included, but were not limited to, change in skin and fur, eyes, mucous membranes, respiratory and circulatory systems, autonomic and central nervous systems, somatomotor activity and behaviour pattern.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A Functional Observational Battery (FOB), including home cage observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations, was performed on all animals once before exposure and at least once per week during pre-breed, mating (both sexes), gestation and lactation (females). Five males and five females per dose group were evaluated for auditory function, motor activity and assessment of grip strength prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights for males were determined and recorded initially and then weekly until termination. The body weights of females were recorded in the same manner until confirmation of mating. During gestation, females were weighed on gestation days 0, 7, 14 and 20. Dams producing litters were weighed on lactation days 0, 4, 7, 14 and 21; body weight gains were computed.

FOOD CONSUMPTION: Yes
- Food consumption for each animal was determined weekly during the pre-breed treatment period. During pregnancy of females, feed consumption was recorded for gd 0-7, 7-14 and 14-20. During lactation of the litters, maternal feed consumption was measured for lactation days 0-4, 4-7, 7-14 and 14-21, although maternal food consumption after day 14 was confounded by the contribution from the pups since the pups were self-feeding by this time. Food consumption was not measured during the cohabitation period since 2 adult animals were in the same age.

OTHER: at the end of the mating period, 5 randomly selected males per group were housed in metabolism cages overnight and urine was collected. The urine samples were observed visually for colour, appearance and total volume. The presence of glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite and leukocytes in urine were evaluated.
Haematology and clinical chemistry parameters were evaluated during the study from randomly selected males and females (5/sex/group). Prior to mating, blood was collected from the tail vein of 5 selected females per group for haematology evaluation. In addition, blood was collected via cardiac puncture at necropsy from randomly selected rats (5 males and 5 females per group, fasted overnight) for haematology (males only) and clinical biochemistry (males and females) evaluation. Clinical chemistry assays for albumin, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, total cholesterol, creatinine, glucose, total protein, and for the electrolytes sodium, potassium, and chloride were performed. The haematology parameters measured included: red blood cell count; total, corrected and differential leukocyte counts; platelet count; haemoglobin concentration; haematocrit; red cell distribution width; mean platelet volume; mean corpuscular volume; mean corpuscular haemoglobin; mean corpuscular haemoglobin concentration; and prothrombin time. Blood smears were prepared for microscopic differentiation of white blood cells and assessment of red blood cell morphology.

Sperm parameters (parental animals):

Parameters examined in male parental animals: The organs weighed and retained from all animals were: epididymides (pair), seminal vesicles with coagulating glands and the fluids (pair) and testes (pair). Full histopathology of the organs was performed for selected high dose and control males and females (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure).

Parameters examined in male offspring: The organs weighed and retained from all animals were: epididymides (pair), seminal vesicles with coagulating glands and the fluids (pair) and testes (pair).

At the time of sacrifice, 1 testis from each adult male was frozen at ~-20 °C for subsequent enumeration of testicular homogenisation-resistant spermatid heads for high dose and control males. In addition, 1 cauda epididymis from each male was immediately removed, weighed and seminal fluid from the cauda was assessed for sperm number, motility and morphology. Sperm motility (motile and progressively motile) was assessed immediately after necropsy for all males; number and morphology were evaluated using appropriately retained sperm samples initially obtained from the high dose and control males.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- The size of each litter was adjusted to 10 by eliminating extra pups by random selection to yield, as nearly as possible, 5 males and 5 females per litter. To provide data on the pnd 4 pups, the pups culled to standardise litters on pnd 4 were euthanised and subjected to a complete gross necropsy, with any gross lesions retained in fixative; non-retained tissues and carcasses from the necropsied pups were discarded.

PARAMETERS EXAMINED
All pups were counted, sexed, and examined as soon as possible after birth to determine the number of viable and stillborn pups from each litter. In addition, all live pups were weighed. Thereafter, litters were evaluated for survival on pnd 4, 7, 14 and at weaning (pnd 21). Individual anogenital distance and body weight were recorded on pnd 0 for all offspring.
All live pups were counted, sexed, weighed individually and examined grossly at birth, pnd 4, 7, 14 and at weaning (pnd 21). The body weights and sexes were recorded on an individual basis, but the pups were not uniquely identified. Anogential distance was recorded with the individual pup weight on pnd 0 for all pups, and the presence or absence of retained nipples and areolae on the ventrum was recorded for male offspring on ~pnd 11 to 13. All pups were examined for physical abnormalities at birth and through the pre-weaning and post-weaning period.
At weaning (pnd 21) at least 1 female and 1 male from each litter, for a total of 10/sex/group were selected on a random basis to continue treatment for at least 7 more weeks. During the post-weaning period, observations for mortality were made twice daily and clinical examinations were conducted and recorded daily until termination. Post-weaning body weights and food consumption measurements were recorded weekly until termination of the selected male and female pups.
Additional post-weaning observations and procedures for each retained female included examination of patency of vaginal opening (from pnd 22 until acquisition of vaginal opening, with body weight recorded on day of acquisition) and determination of oestrous cyclicity and normality, evaluated by vaginal smears taken daily the last 3 weeks of the post-wean exposure period prior to scheduled sacrifice. For each retained male offspring, observations for the cleavage of the balano-prepreputial gland (PPS) began at 35 days of age and continued until acquisition of PPS, with body weight recorded on the day of acquisition.
FOBs, including home cage observations, handling observation, open field observation, sensor and neuromuscular observations and physiological observations were performed on 5 males and 5 males per group once midway during the post-wean exposure period. Grip strength was also assessed for the 5 males and 5 females per group selected for FOB during the last week of the post-weaning exposure period.
Five selected males per group were singly housed in metabolism cages overnight prior to necropsy for collection of urine for urinalysis. The assays were the same as those performed for the parental males. At necropsy, blood was collected from 5 males and 5 females per group (fasted overnight) for haematology and clinical biochemistry. Assays were performed as they were for the parental animals.

GROSS EXAMINATION OF DEAD PUPS:
All pups dying during lactation were necropsied, when possible, to investigate the cause of death. No organs from these pups were weighed or saved.

Postmortem examinations (parental animals):

All parental animals (including recovery animals) in all groups were subjected to a complete gross necropsy, where selected organs were weighed and/or retained in fixative for possible subsequent histopathologic evaluation. Uterine nidation scars were also recorded for the females. The organs weighed and retained from all animals were: epididymides (pair), ovaries (pair), prostate, seminal vesicles with coagulating glands and the fluids (pair), testes (pair), uterus with cervix and vagina.
In addition, the following organs were weighed and saved from 5 randomly selected males and females per group: adrenal (pair), brain (including cerebrum, cerebellum and pons) heart, kidneys (pair), liver, spleen and thymus. In addition to the organs listed above, the organs listed below were also saved from the same randomly selected animals: all gross lesions, bone marrow (femur), lymph nodes (1 cervical near route of administration and 1 mesenteric distant from the route of administration), peripheral nerve (sciatic nerve), small and large intestines (including Peyer’s patches), spinal cord, stomach, thyroid, trachea and lungs (preserved by inflation with fixative and them immersion fixed) and urinary bladder.
Organ weights were reported as absolute and relative to terminal body weight and brain weight.

Full histopathology of the organs listed above was performed for the selected high dose and control males and females (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure). In addition, based on histopathology findings in the kidneys from the high dose males, histopathology was performed on the kidneys from the low and mid dose males (5/group), as well as the control and high dose recovery males (5/group).

The fixed uteri from any females failing to produce a litter were stained for confirmation of pregnancy. This staining procedure did not interfere with subsequent histopathologic evaluation.

Postmortem examinations (offspring):

All retained animals were subjected to a complete gross necropsy, where selected organs were weighed and/or retained in fixative for possible subsequent histopathologic evaluation. The organs weighed and retained from all animals were: epididymides (pair), ovaries (pair), prostate, seminal vesicles with coagulating glands and the fluids (pair), testes (pair), uterus with cervix and vagina.
In addition, the following organs were weighed and saved from 5 randomly selected males and females per group: adrenal (pair), brain (including cerebrum, cerebellum and pons), heart, kidneys (pair), liver, spleen and thymus. In addition to these organs the following were also saved from the same randomly selected animals: all gross lesions, bone marrow (femur), lymph nodes (1 cervical near route of administration and 1 mesenteric distant from the route of administration), peripheral nerve (sciatic nerve), small and large intestines (including Peyer’s patches), spinal cord, stomach, thyroid, trachea and lungs (preserved by inflation with fixative and them immersion fixed) and urinary bladder.
The following organs were weighed from the non-selected F1 weanlings subjected to necropsy: brain, epididymides (pair), ovaries (pair), spleen, testes (pair), thymus and uterus (with cervix and vagina).
Organ weights were reported as absolute and relative to terminal body weight and brain weight.

Full histopathology of the organs listed as performed for the selected high dose and control males and females. In addition, based on histopathology findings in the kidneys from the high dose parental males, histopathology was performed on the kidneys from the pups in the low and mid dose males (5/group).

Statistics:

See below

Reproductive indices:

FEMALES
Mating index (%): (# females sperm positive/# females paired) x 100
Fertility index (%): (# females pregnant/# females sperm positive) x 100
Gestational index (%): (# females with live litters/ # females pregnant) x 100

MALES
Mating index (%): (# males impregnating females/# males paired) x 10
Fertility index (%): (# males siring litters/# males impregnating females) x 100
Pregnancy index (%): (# pregnant females/ # males impregnating females) x 100

Offspring viability indices:

- Live birth index (%): (# live pups at birth/total # of pups born) x 100
- 4-day survival index (%): (# pups surviving 4 days (pre-cull)/total # live pups at birth) x 100
- 7-day survival index (%): (# pups surviving 7 days/ total # live pups at 4 days (pre-cull)) x 100
- 14-day survival index (%): (# pups surviving 14 days/total # live pups at 7 days) x 100
- 21-day survival index (%): (# pups surviving 21 days/total # live pups at 14 days) x 100
- Lactation index (%): (# pups surviving 21 days/total # live pups at 4 days (pre-cull)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical observations included: rooting in the bedding post dosing which occurred in 3, 3, and 10 males at 0, 50, and 300 mg/kg/day, respectively: and 1 male in the control group and 6 males in the high dose group exhibited salivation pre-dosing.
Clinical signs in the recovery males included rooting post-dosing in all males at 300 mg/kg/day and efflux of the dosing formulation, and/or salivating prior to and/or post-dosing in 1 male each at 300 mg/kg/day during the dosing period. No other treatment related signs were recorded during the dosing or recovery periods.
Clinical signs related to treatment in the females included rooting in the bedding post-dosing in 3 of 10 females at 50 mg/kg/day and 9 of 10 females at 300 mg/kg/day. Salivation pre-dosing and/or post-dosing occurred in 4/10 females at 300 mg/kg/day.
Clinical observation of females during gestation included rooting in the bedding post-dosing that occurred in 2, 1, 5 and 8 females at 0, 5, 50 and 300 mg/kg/day, respectively. Salivation prior to dosing occurred in 1 female at 50 mg/kg/day and 4 females at 300 mg/kg/day. No other clinical signs were considered to be related to treatment.
Maternal clinical observations during lactation included rooting in the bedding post-dosing (6 females) and salivation prior to dosing (4 females) at 300 mg/kg/day.

Mortality:

no mortality observed

Description (incidence):

No males (including recovery) or females died on study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant differences among groups for male body weight. However, body weight changes for sd 0-7, 14-21 and 0-28 were significantly decreased at 300 mg/kg/day. Body weight change was also significantly reduced at 50 mg/kg/day but only for sd 14-21. At scheduled sacrifice mean body weights showed a dose related decreasing trend, but no pairwise comparisons were statistically significant.
High dose recovery males exhibited significantly reduced body weight during the 4-week treatment period (sd 7, 14, 21 and 28) and during the 2-week recovery period (sd 35 and 42). Body weight gain was significantly reduced in the high dose recovery males for sd 0-7, 7-14 and 0-28 during the 28-day dosing period, and was unaffected during the 2-week recovery period. Sacrifice weight was significantly reduced in the high dose group compared to the control value.
There was a significant reduction in female body weight on sd 14 at 50 mg/kg/day and significantly reduced body weight changes at 50 and 300 mg/kg/day for sd 0-7 and for the premating period (sd 0-14). None of these changes showed a dose response, however. In addition, body weight was significantly decreased at the time of FOB measurement at 50 and 300 mg/kg/day in weeks 3, 4 and 5 (although again without a dose response), and at 300 mg/kg/day in week 6.
There was a significant decrease in the maternal body weight on gd 7 and 20 (but not on gd 14) at 300 mg/kg/day. Significant decrease in body weight change occurred at 300 mg/kg/day for gd 14-20.
There were no significant differences in maternal lactation body weights at 0, 5, 50 ro 300 mg/kg/day.
At scheduled sacrifice mean body weights of the females were equivalent across all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no significant effects on feed consumption expressed as g/day for males. However, there was a significant reduction on male feed consumption expressed in g/kg/day for sd 0-7 at 300 mg/kg/day.
Ffor the females, feed consumption, expressed as g/day for the first week of pre-breed and for the entire pre-breed period (sd 0-14), was significantly decreased at 300 mg/kg/day. When feed consumption was expressed as g/kg/day, there were significant reductions at 50 and 300 mg/kg/day for sd 0-7 and a significant reduction at 300 mg/kg/day for sd 0-14. There were no significant effects on feed consumption (expressed as g/day or g/kg/day) in any group during the second week of the pre-breeding period (sd 7-14).
There were no treatment related effects across groups for maternal feed consumption when expressed as g/day or g/kg/day for d 0-7, 7-14, 14-20 or 0-20.
Maternal lactational feed consumption (g/day and g/kg/day) was unaffected at 0, 5, 50 and 300 mg/kg/day for pnd 0, 4, 7, 14 and 21.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology performed prior to mating in the females indicated that red blood cell distribution width and percent of segmented neutrophils were significantly reduced at 300 mg/kg/day. These were not considered related to treatment because of the lack of effects in associated blood parameters. All other haematology and prothrombin time values in females were unaffected across all dose groups.
No effects were observed in males prior to sacrifice.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No effects were observed in males or females prior to sacrifice.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No effects were observed in males prior to sacrifice.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

FOB was performed once during quarantine for all animals as a base line. There were no significant differences for home-cage observations, handling observations, sensory and neuromuscular observations, or open field observations in males or females assigned to any dose group.
There were no treatment-related changes in any of the FOB tests conducted weekly through pre-breeding and breeding in the males. Statistically significant differences were not dose related, showed no pattern consistent with a toxic effect, and were not considered treatment related. There were no effects of treatment on motor activity, auditory startle or grip strength which were performed in week 4.
There was no effect of treatment on FOB in the females during the pre-breed period or during weeks 3 and 4 (2-week mating period). Average hindlimb grip strength was significantly reduced at 300 mg/kg/day for weeks 5, 7 and 8. No other statistically significant differences were considered to be related to treatment based upon a lack of correlation with other observations/measurements, lack of dose response, consistency across measurement period, and/or inconsistency with toxicologically relevant effects.
Five females per group that littered were selected for auditory startle, motor activity, and grip strength testing during the last week of lactation (pnd 14-21). No effect of treatment was seen on average force of jump or average duration of jump on any blocks tested during auditory startle. No effects were seen for motor activity or for average forelimb or hindlimb grip strength.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment related histopathological finings in males included 4 of 5 males with renal tubular necrosis and 5 of 5 males with diffuse renal tubule regeneration at 300 mg/kg/day. Other histological findings were considered to be typical for this age and strain of rat. Since the renal tubular necrosis and tubular regeneration were considered treatment related at 300 mg/kg/day, kidneys for the parental and F1 generation in the mid and low dose groups and the kidneys from the recovery males were also submitted for histopathological evaluation. No finings considered related to treatment were observe in the mid and low dose groups.
In the recovery males, of the 5 males each in the control and high dose groups for which kidneys were submitted, 3 males at 300 mg/kg/day had diffuse tubular regeneration, considered to be associated with the treatment related lesions observed in the animals sacrificed following dosing. The incidence and severity of these lesions were reduced from those following treatment. Tubular necrosis was not evident by the end of the recovery period. No other lesions were considered to be related to the earlier treatment.
In the females there were no treatment related histopathological lesions. Recorded findings occurred in the control and treatment groups and/or were not dose related and, therefore, were not considered a result of test substance toxicity.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

In the males, relative (to body weight) paired testes weight at 50 and 300 mg/kg/day and paired epididymis weight at 300 mg/kg/day were significantly increased. These differences were considered to be related to the slightly reduced body weights because there was no effect on the absolute weights.
In the recovery males, brain and paired testes weights, relative to terminal body weight, were significantly increased at 300 mg/kg/day (most likely due to reduced body weights at this dose).

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The number of sperm-positive pregnant females was 9, 9, 9 and 8 for 0, 5, 50 and 300 mg/kg/day, respectively.
There were no significant effects of exposure on mating, fertility, or pregnancy indices during the production of the F1 offspring. Pre-coital interval and gestational length were unaffected by treatment.

There were no significant differences across groups for percent post implantation loss per litter, number of live pups on pnd 0, or the number of dead pups at birth (pnd 0). The live birth index, the pnd 4, 7, 14 and 21 day survival indices, as well as the lactation index (pnd 4 and post-cull 21) were equivalent across all dose groups.

Although there was a significant decrease in the number of total implantation sites per litter at 300 mg/kg/day, the lack of associated effects on other reproductive parameters do not allow for a direct conclusion that this observation was a direct effect on reproduction.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects at this dose level

2

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

3

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects at this dose level

4

Dose descriptor:

LOEL

Remarks:

reproduction

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related clinical signs observed during the lactational period.
Clinical observations in adult males included, rooting in bedding pre or post dosing (2, 7 and 11 at 5, 50 and 300 mg/kg/day, respectively) and salivating prior to dosing (5 at 300 mg/kg/day). No other treatment related observations were recorded.
Clinical observation in adult females included alopecia in 1 female each at 0 and 50 mg/kg/day, efflux of doing formulation in 1 female at 5 mg/kg/day, rooting in the bedding post dosing in 1, 6 and 10 females at 0, 50 and 300 mg/kg/day, respectively and salivating prior to dosing in 10 females at 300 mg/kg/day.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were 9, 10, 10 and 9 live litters on pnd 0 at 0, 5, 50 and 300 mg/kg/day, respectively. The mean number of live pups per litter for pnd 0, 4, 7, 14 and 21 was unaffected across all groups. There was no treatment-related mortality during the lactational period.
3 adult male deaths occurred prior to scheduled sacrifice (2 in the control group and 1 at 5 mg/kg/day) and were considered to be due to dosing errors.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup body weights per litter (sexes combined or separately) were unaffected by treatment on pnd 0, 4, 7, 14 and 21 across all dose groups.
There were no effects on male or female body weights per litter at sacrifice on pnd 21.
Adult male body weights were equivalent across all groups during the post-weaning period except for a significant reduction at 300 mg/kg/day for pnd 57. Body weight changes during the post-weaning period included significant decreases in body weight for pnd 50-57 at 5 and 300 mg/kg/day and significant increases in body weight for pnd 71-78 at 50 and 300 mg/kg/day. No other effect on body weight or body with change was observed for the pnd 28-78 period. The values for pnd 78-85 were for 1-5 males/group, so no statistical analyses were performed. Overall there were no treatment related effects on body weight. At schedule sacrifice, at ~77 days of age, mean body weights were equivalent across all groups.
In the adult females, there were no significant differences in body weights on pnd 22-85 across all dose groups. Body weight change showed a transient significant decrease only for pnd 71-78 at 50 mg/kg/day. At scheduled sacrifice, at ~85 days of age, mean adult female body weights were equivalent across all groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was a decrease in food consumption of the adult males when expressed as g/day only on pnd 43-50 at 5 mg/kg/day, with no effects at 50 or 300 mg/kg/day. Feed consumption expressed as g/kg/day was significantly increased for pnd 36-43, pnd 64-71, pnd 71-78 and pnd 22-78 at 300 mg/kg/day, with no effects at 5 or 50 mg/kg/day. None of these differences were considered treatment related.
Except for 2 non-dose related reductions in feed consumption at 5 and 50 mg/kg/day (pnd 43-50 and 50-57, respectively), feed consumption values (expressed as g/kg/day) were equivalent across all dose groups for the adult females from pnd 22-78. Feed consumption values (expressed as g/day) were significantly increased only for pnd 71-78 and only at 300 mg/kg/day.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Description (incidence and severity):

Male age at acquisition of PPS (preputial separation) was equivalent across all dose groups (40.8, 40.0, 41.5, and 41.7 days at 0, 5, 50 and 300 mg/kg/day, respectively). For the adult males, there were no significant differences among groups for percent motile sperm or for progressively motile sperm evaluated in all groups. Epididymal sperm concentration, spermatid head concentration, daily sperm production per testis, efficacy of daily sperm production and percent abnormal sperm were equivalent in the control and high dose groups (these parameters were not assessed at 5 or 50 mg/kg/day).
Adult female age at acquisition of VP (vaginal patency) was equivalent at 0, 5, 50 and 300 mg/kg/day (30.9, 31.3, 31.7 and 31.6 days, respectively). The oestrous cycle lengths for the females were equivalent across all dose groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on male organ weights when expressed as absolute, relative to sacrifice weight or relative to brain weight. In females, paired ovary weights when expressed as absolute and relative to sacrifice weight and relative to brain weight, were significantly reduced at 5 and 50 mg/kg/day but not at 300 mg/kg/day. Due to the lack of dose response, these differences were not considered to be related to treatment.
In the adult males, absolute and relative weight of the spleen (expressed as relative to body weight and brain weight) were significantly decreased at all doses. Seminal vesicles with coagulating gland weights, relative to brain weight, were significantly increased at 5 mg/kg/day but the difference was not considered to be treatment related. No other absolute or relative organ weights were significantly different from controls.
In adult females, absolute organ weights and organ weights relative to terminal body and brain weights were equivalent across all groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related findings at necropsy of the pnd 4 culled pups. There were 34, 38, 39 and 32 male offspring and 36, 41, 40 and 37 female offspring evaluated at schedule sacrifice on pnd 21 at 0, 5, 50 and 300 mg/kg/day, respectively. There were no treatment related necropsy findings for pups on pnd 21.
There were no treatment related gross necropsy findings in the adult males or females.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related histopathological findings in the adult males or females.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Mean female and male anogenital distances (absolute) per litter on pnd 0 were equivalent across dose groups. Anogenital distance adjusted for body weight was unaffected in males but was significantly increased in females at 50 mg/kg/day. This difference was not considered to be related to treatment due to the lack of dose response and the minimal magnitude of the change.
The sex ratio (percent male pups/litter) was unaffected at any dose for any lactational interval. The average number of areolae per male pup and the percent male pups with 1 or more areolae was significantly increased at 50 mg/kg/day. This difference was not considered to be related to treatment due to the lack of dose response and the minimal magnitude of the change.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

FOB was performed once midway through the post-weaning period. In the adult males there were no significant differences for home cage observations, handling observations, sensory and neuromuscular observation, or open field observation in males assigned to any dose group. There was also no treatment related effect on average forelimb or hindlimb grip strength in any dose group.
In the adult females there were no significant differences for home cage observation, handling observations, sensory and neuromuscular observation, or open field observations in females assigned to any dose group, and there were no effects on average forelimb or hindlimb grip strength in the adult females at any dose level.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 300 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effects were seen in the F1 generation.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Summary of F1 Adult Systemic Toxicity - Key Parameters and Statistically Significant Differences

p-Cumylphenol (mg/kg/day)	F1
	0	5	50	300
F1 ADULT MALES
Deaths	2	1	0	0
Postweaning Period (pnd 22 to 78)
Body Weights	---	---	---	d*
Weight Change	---	d*	uu*	u,dd*
Feed Consumption:                g/day	---	d*	---	---
g/kg/day	---	---	---	u,uu*
Hematology
Mean Corpuscular Volume	---	---	uuu	---
Mean Corpuscular Hemoglobin	---	---	---	d
Mean Corpuscular Hemoglobin Concentration	---	dd	d	ddd
Necropsy**
Spleen                                                  A	---	ddd	ddd	ddd
R - Body	---	dd	dd	dd
R - Brain	---	dd	ddd	dd
Seminal Vesicles                       A	---	---	---	---
R - Body	---	---	---	---
R - Brain	---	uu	---	---
F1 ADULT FEMALES
Deaths	0	0	0	0
Postweaning Exposure (pnd 22 to 78)
Weight Change	---	---	d*	---
Feed Consumption:                   g/day	---	---	^---	u*
g/kg/day	---	dd*	d*	---

*Different levels of significance or no statistical significance noted for different intervals within this parameter

**Histopathology performed on control and high dose F1 males and females and for F1 males only at 5 and 50 mg/kg/day

u, uu, uuu = statistically significant increase; 1 symbol - p is less than 0.05; 2 symbols - p is less than 0.01; 3 symbols - p is less than 0.001

d, dd, ddd = statistically significant decrease; 1 symbol - p is less than 0.05; 2 symbols - p is less than 0.01; 3 symbols - p is less than 0.001

--- = no statistically significant difference

A = absolute organ weight in grams

R - Body = organ weight relative to terminal body weight (%)

R - Brain = organ weight relative to terminal brain weight (%)

Table 2: Summary of F0 Parental Male and Female Reproductive Toxicity

p-Cumylphenol (mg/kg/day)	F0
	0	5	50	300
FEMALES
Precoital interval, days	2.5	2.1	3.0	2.9
Indices:              Mating	---	---	---	---
Fertility	---	---	---	---
Gestational	100.0	100.0	100.0	100.0
Gestational length, days	22.1	22.3	22.2	21.9
No. maternal deaths on gd 21 - pnd 6	0	0	0	0
No. implant sites/litter	15.78	15.50	13.90	13.11**
% postimplantation loss/litter	5.59	8.44	2.75	3.04
No. total pups/litter, pnd 0	15.0	14.5	13.6	12.9
No. live pups/litter, pnd 0	15.0	14.1	13.5	12.9
No. dead pups/litter, pnd 0	0.0	0.4	0.1	0.0
No. females pregnant	9	10	10	9
No. litters on pnd 0	9	10	10	9
No. litters on pnd 21	9	10	10	9
MALES
Indices:              Mating	---	---	---	---
Fertility	---	---	---	---
Pregnancy	---	---	---	---

--- = no statistically significant difference

** = p is less than 0.01

Table 3: Summary of F1 Offspring Toxicity

p-Cumylphenol (mg/kg/day)	F1
	0	5	50	300
Stillbirth index	0.0	2.9	0.8	0.0
Live birth index	100.0	97.1	99.2	100.0
Average number of areolae per male pup	0.77	0.90	2.04*	1.07
Weanling Necropsy (pnd 21)
Males
Body weight (g)	---	---	---	---
Females
Body weight (g)	---	---	---	---
Paired Ovaries                                      A	---	dd	dd	---
R - Body	---	ddd	d	---
R - Brain	---	dd	dd	---
Weanling Necropsy Gross Findings
Male:  Kidney hydronephrosis,right†	0	NA	1	0

†Indicated parameters were assessed in control, mid-, and high-dose animals only

NA = not applicable

u, uu, uuu = statistically significant increase; 1 symbol - p is less than 0.05; 2 symbols - p is less than 0.01; 3 symbols - p is less than 0.001

d, dd, ddd = statistically significant decrease; 1 symbol - p is less than 0.05; 2 symbols - p is less than 0.01; 3 symbols - p is less than 0.001

--- = no statistically significant difference

* = statistically significantly different from control group value at p is less than 0.05.

A = absolute organ weight in grams

R - Body = organ weight relative to terminal body weight (%)

R - Brain = organ weight relative to terminal brain weight (%)

Applicant's summary and conclusion

Conclusions:

The NOAEL for the parental rats was determined to be 50 mg/kg/day and the NOAEL for the F1 generation was determined to be 300 mg/kg/day.

Executive summary:

The repeated dose oral toxicity was investigated per OECD Test Guideline 422 under GLP conditions. The study exceeded the guideline by following the F1 offspring to adulthood and includes an assessment of neurologic function.

P-cumylphenol, administered by gavage once daily at 0, 5, 50 and 300 mg/kg/day to parental Sprague-Dawley rats (10/sex/group) by gavage in corn oil through pre-breed, mating, gestation and lactation and direct dosing to F1 offspring from weaning to scheduled sacrifice resulted in systemic toxicity (manifested as renal tubular necrosis) at 300 mg/kg/day in the parental males. The LOAEL was determined to be 300 mg/kg/day for parental males. A slight decrease in uterine implantations at 300 mg/kg/day in the parental females was noted; however, based on the lack of other associated effects on reproductive parameters and the screening nature of this study a LOEL for females was established at 300 mg/kg/day. No toxicity in the F1 offspring from birth through 7 weeks of post-weaning dosing were observed.

The NOAEL for the Parental rats was determined to be 50 mg/kg/day and the NOAEL for the F1 generation was determined to be 300 mg/kg/day.