p-cymene

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-16 to 2019-03-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

Deviations were not considered to have compromised the validity or integrity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD®(SD)

Details on species / strain selection:

OECD Test Guideline 422 was designed for use with the rat. Dosing studies in a rodent species are required by chemical regulatory agencies such as ECHA and US EPA. In addition, a historical control data base is available for comparative evaluation.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, North Carolina 27610)
- Females (if applicable) nulliparous and non-pregnant: Not Specified
- Age at study initiation: Approximately 11 to 13 weeks
- Weight at study initiation: Males: 332 - 434 grams; Females: 235 - 299 grams
- Fasting period before study: Not specified
- Housing: Polycarbonate cages with a stainless steel mesh lid. From arrival until one day prior to treatment, animals were pair or group housed (2 or 3 rats of the same sex per cage, respectively) in solid bottom cages with cellulose-based contact bedding. From the initiation of treatment (pre-cohabitation phase until termination of the study), the P0 males and females were pairhoused [same sex and treatment group per cage (except during the cohabitation phase)] in suspended, solid bottom cages with cellulose-based contact bedding. Per the OECD 422 guideline, each P0 female wasindividually housed during presumed gestation and housed with her litter after delivery. During cohabitation, the male and female rats were co-housed (1:1) within each treatment group in suspended, solid bottom cages with cellulose-based contact bedding.
- Diet (e.g. ad libitum): Teklad Global 18% Protein Rodent Diet (Certified), 2018C (Envigo, Madison, Wisconsin) ad libitum. Fresh feed was presented weekly in the home cage of each animal.
- Water (e.g. ad libitum): Potable water from the public supply (New Jersey-American Water Company, Cherry Hill, New Jersey) via an automated watering system. ad libitum.
- Acclimation period: 19 days (The pre-test period was 19 days. All animals were examined during the pre-test period to confirm suitability for study. Pre-test procedures other than routine husbandry care and identification procedures were not performed until animals had been allowed to stabilize for at least 5 days. Prior to assignment to study, all animals were examined to ascertain suitability for study).

DETAILS OF FOOD AND WATER QUALITY:
Teklad Global 18% Protein Rodent Diet (Certified), 2018C (Envigo, Madison, Wisconsin) was provided without restriction. Fresh feed was presented weekly in the home cage of each animal. Analysis of each feed lot used during this study was performed by the manufacturer. There were no known contaminants in the feed that were expected to interfere with the results of this study. Potable water from the public supply (New Jersey-American Water Company, Cherry Hill, New Jersey) via an automated watering system was provided without restriction. Water analyses are conducted by New Jersey-American Water Company, Cherry Hill, New Jersey (Raritan-Millstone Plant) to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations (40 CFR Part 141). In addition, water samples are collected biannually from representative rooms in the Testing Facility; chemical and microbiological water analyses are conducted on these samples by a subcontract laboratory. There were no known contaminants in the water which were expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26°C
- Humidity (%): 30 to 70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): A twelve hour light/dark cycle was provided and controlled via an automatic timer

IN-LIFE DATES: From: 2018-05-17 & 2018-05-24 To: 2018-08-04

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
p-Cymene was prepared for administration by mixing appropriate amounts of the test item with the vehicle to achieve the desired concentrations (details provided in Table 1). The test item was used as supplied when calculating quantities to be used during dose preparation. Fresh formulations were prepared once weekly and stored at room temperat ure (20± 5 °C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (Justification not specified)
- Concentration in vehicle: 0, 10, 20, & 40 mg/L for control, low-, mid-, and high dose levels, respectively.
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required): Spectrum Chemical Mfg. Corp (755 Jersey Avenue, New Brunswick, New Jersey 08901); Lot# 2HB0068
- Purity: Assume 100%

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in the vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
In the absence of positive signs of mating, the morning of the last day of cohabitation was considered “presumed GD0” for that female. If the presumed pregnant dam did not appear visibly/palpably pregnant, she was euthanized on presumed GD 25. If a female showed no positive evidence of mating, but appearedvisibly/palpably pregnant, it was allowed to deliver, and the estimated GD 0 calculated.
- After successful mating each pregnant female was caged (how): Once mated, the female rat was removed from the mating cage and housed individually until delivery

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity:
Prior to initiation of dosing, homogeneity of dose formulations was demonstrated by taking two samples each from the top, middle and bottom portion (2.5 mL/sample) of the low and high concentration formulations prepared for use on the study.

Stability:
Stability of the low- and high-concentration dose formulations under the storage conditions used in this study was determined at time points of 4 and 8 days from preparation under the method validation Study No. TN40YX.

Dose Concentration:
Samples collected from the middle of the dose formulations for homogeneity analysis were used for the dose confirmation results of the low and high concentrations. Two samples (2.5 mL each) were taken from the middle region of each formulation (including control) on the day of the first and last dose preparation. One sample was analyzed, in duplicate, for dose confirmation analysis and one sample was retained at room temperature (20 ± 5°C). Retained samples were discarded after valid analytical results are obtained.

Method of Analysis: Analyses were performed by the Department of Formulation and Inhalation Analysis at the Testing Facility.

Duration of treatment / exposure:

P0 males: 2 weeks pre-cohabitation, during cohabitation (up to 2 weeks) and continuing during post-cohabitation until the day prior to termination (approximately 35 days).

P0 females: 2 weeks pre-cohabitation, cohabitation (up to 2 weeks) and during gestation and lactation continuing until LD 13 (approximately 63 days).

Frequency of treatment:

Once Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Low Concentration

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Medium Concentration

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

High Concentration

No. of animals per sex per dose:

10 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses selected for the study were based on a 14-day repeat dose oral (gavage) range-finding toxicity study (Envigo Study No. XD46CR). One 500 mg/Kg/day female was sacrificed as moribund onStudy Day 13. This animal was thin and exhibited rapid breathing, decreased activity and hunched posture. There were no macroscopic findings indicative of a gavage accident. There were no adverse clinical signs at any dose level for both genders except for the moribund 500 mg/Kg/day female animal. Body weights, body weight changes and food consumption were significantly reduced in the 500 mg/Kg/day group. Body weights, body weight changes and food consumption were also reduce d in the 150 mg/Kg/day group. Necropsy and gross pathology revealed one 150 mg/Kg/day male with discoloured lungs and bronchi (dark red area on the right caudal lobe [
The high dose (200 mg/Kg/day) used in this OECD TG 422 study is 40% of the high dose level in the 14-day oral gavage range-finding toxicity study. It was anticipated that the 200 mg/Kg/day dose level would not cause mortality but might cause some adverse toxicity. The low dose (50mg/Kg/day) is 25% of the high dose in the 14-day oral gavage range-finding toxicity study and was anticipated to cause little or no toxicity. The mid-dose (100 mg/Kg/day) is 50% of the high dose.

- Rationale for animal assignment (if not random):
Males considered suitable for study on the basis of pre-test physical examinations, body weight data and any other pre-test evaluations, were randomly assigned to control, treatment or spare groups, using a computerized program, in an attempt to equalize mean group body weights. Individual weights of male animals placed on test were within ± 20% of the mean weight. Females considered suitable for study on the basis of pre-test physical examinations, body weight data, regular estrous cycles and any other pre-testevaluations, were randomly assigned to control, treatment or spare groups, using a computerized program, in an attempt to equalize mean group body weights. Normally cycling P0 females (demonstrating 4 to 5 days cyclicity) were selected for each dose group. Individual weights of female animals placed on test were within ± 20% of the mean weight.

- Fasting period before blood sampling for clinical biochemistry: Yes

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed in their cages at least twice daily for mortality and general condition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: P0 male rats were removed from their cages and observed once weekly (prior to dosing) from the initiation of treatment until termination. P0 female rats were removed from their cages and observed once weekly (prior to dosing) from the initiation of treatment through mating. Once mating was confirmed, animals were observed on GD 0, 7, 14 and 20 and female rats that delivered a litter were observed on LD 1, 4, 7 and 14. Maternal behavior was observed daily from GD 18 to LD 13. Female rats without evidence of mating from the male pairing were moved into the gestation phase at the end of the cohabitation phase and upon delivery were moved into the lactation phase and examined similarly as other females with evidence of mating. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluation of respiration. During the treatment period, all animals were observed for signs of toxic effects once daily within 2 hours (± 30 minutes) after test material administration

BODY WEIGHT: Yes
- Time schedule for examinations:
P0 male rats: recorded weekly (prior to dosing), from the initiation of treatment and weekly thereafter throughout the study and at termination.

P0 female rats: recorded weekly (prior to dosing), from the initiation of treatment and weekly during the pre-cohabitation and cohabitation phases until mated. Mated female rats were weighed on GD0, 7, 14 and 20 and female rats that delivered litters were weighed on LD 1, 4, 7 and 13 and at termination on Day 14. Female rats without evidence of mating from the male pairing were moved into the gestation phase and upon delivery were moved into the lactation phase and weighed similarly as other females with evidence of mating.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption for the male and female rats was measured (weighed) weekly prior to initiation of treatment (pre-test) and during the pre-cohabitation phase. Food consumption was not measured during the cohabitation phase when male rats were being co-housed with female rats. For male rats, food consumption was measured weekly during the post-cohabitation phase. For female rats, gestation and lactation food consumption was measured on GDs 0-7, 7-14 and 14-20 and on LDs 1-7 and 7-14, respectively.Food consumption for the female rats without evidence of mating was measured similarly as other females with evidence of mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was obtained from anesthetized P0 rats as a terminal procedure via puncture of the vena cava. P0 males were terminated on Day 35 (approximately) while P0 females were terminated on Day 63 (approximately).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (Animals were fasted overnight prior to collection)
- How many animals: Hematology samples were collected from the first 5 surviving animals/sex/group. Coagulation samples were collected from upto the second
5 surviving animals/sex/group.
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was obtained from anesthetized P0 rats as a terminal procedure via puncture of the vena cava. P0 males were terminated on Day 35 (approximately) while P0 females were terminated on Day 63 (approximately).
- Animals fasted: Yes (Animals were fasted overnight prior to collection)
- How many animals: Clinical chemistry samples were collected from the first 5 surviving animals/sex/group.
- Parameters checked in table [No.3] were examined.

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity, grip strength and locomotor activity were performed prior to dosing during Week 5 for the first five P0 males in each group and on LD 8 ± 1 for the first five P0 females.
- Dose groups that were examined: All dose groups
- Battery of functions tested: sensory activity, grip strength, motor activity

IMMUNOLOGY: No

OTHER:
Thyroid Hormone Analysis: P0 males and females at termination (Up to 10 animals/sex/group). Blood for thyroid hormone analysiswas collected into polypropylene serum gel tubes with no anticoagulant and allowed to clot for at least 30 minutes at room temperature. Serum was harvested within 1 hour after collection of each blood sample. Serum was separated by centrifugation (for 10 minutes at approximately 2000 g, at approximately 2 to 8°C). Approximately 255 µL or more of serum was harvested and stored frozen (-80 ° ± 10 °C) at the Testing Facility until shipped for analysis. Serum samples from P0 males were assessed for serum levels of Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) by AntechGLP. Serum samples (T4) from P0 females were maintained frozen (-80ºC ± 10 ºC) at the Testing Facility pending sample analysis or final disposition of samples.

Oestrous cyclicity (parental animals):

Vaginal smears were obtained daily from each P0 female pre-test(to determine suitability for study)and during the pre-cohabitation and cohabitation phases until mating was confirmed and the stage of estrous was determined.

Sperm parameters (parental animals):

Parameters listed in Tables 4 & 5 were examined in male parental generations:
During the microscopic examination of the testes, special emphasis was placed on the stage of spermatogenesis and the interstitial testicular cell structure. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Not specified

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Litters were observed as soon as possible after completion of parturition for the number of live and dead pups, runts and pup abnormalities and the sex of each pup. Thereafter, litters were observed twice daily (morning and afternoon); all pups in the litter were uniquely identified by tattoo after parturition was completed. Because the litters were not culled, the full number of pups remained in each litter. The presence of dead pups was recorded and these were removed from the litter as found and necropsied. Unusual observations and the absence of milk in the stomach were noted.

Each pup was given a physical examination daily from PND 1 through PND 13. Observations of dead or missing pups were noted. Individual pup body weights were recorded on PND 1, 4, 7, 11 and 13. The sex of each pup was identified on PND 1 and verified on PND 4, 7 and 13.

Anogenital distance was measured in millimeters using a caliper for all pups on PND 4. Nipple retention was assessed for all F1male pups on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
Yes, The presence of dead pups was recorded and these were removed from the litter as found and necropsied.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Thyroid Hormone Analysis – F1 Pups

F1 pups at PND 4 - Up to 2 females per litter(when possible) pooled sample by litter
Pooled blood (~0.6 mL) was obtained from two (when possible) non-fasted but anesthetized (intraperitoneal injection of sodium pentobarbital) female PND 4 F1pups per litter via the greater vessels as a terminal procedure immediately after the appropriate level of anesthesia was reached.

F1pups at PND 13 - Up to 2 males and 2 females(when possible) per litter
Blood (~0.6 mL) was obtained from non-fasted but anesthetized (intraperitoneal injection of sodium pentobarbital) PND 13 F1pups via the greater vessels as a terminal procedure immediately after the appropriate level of anesthesia was reached.

Blood for thyroid hormone analysiswas collected into polypropylene serum gel tubes with no anticoagulant and allowed to clot for at least 30 minutes at room temperature. Serum was harvestedwithin 1 hour after collection of each blood sample. Serum was separated by centrifugation (for 10 minutes at approximately 2000 g, at approximately 2 to 8°C). Approximately 255 µL or more of serum was harvested and stored frozen (-80 ° ± 10 °C) at the Testing Facility until shipped for analysis.

Serum samples for T4 and TSH analysis from PND 13 F1pups were shipped frozen, on dry ice to AntechGLP for analysis. Serum samples (T4) from PND 4
F1 pups were maintained frozen (-80ºC ± 10 ºC) at the Testing Facility pending sample analysis or final disposition of samples.

Postmortem examinations (parental animals):

SACRIFICE
Adult males and females were euthanized by exsanguination following isoflurane inhalation. Necropsy was performed on 10 P0 males/group after males had been treated for 5 weeks. Necropsy schedules were established to ensure that approximately equal numbers of males from each group were examined at similar times of the day. Necropsy was performed on up to 10 P0 females in Groups 1, 2 and 3 on LD 14. Female rats that failed to deliver a litter were euthanized on GD 25.

GROSS PATHOLOGY: Yes (see Table 4 and Table 5)
Macroscopic postmortem examinations were performed on all P0 rats. Postmortem examinations included an external examination as well as a detailed internal examination. Special attention was paid to the organs of the reproductive system. The number of implantation sites, scars and corpora lutea was recorded for each female rat. For apparently non-pregnant animals, and for apparently empty horns, the number of uterine implantation sites were checked after staining with ammonium sulfide.
The first five surviving adult males and lactating females that delivered a litter in each group had organs in Table 4 weighed and the second set of up to five surviving adult males and lactating females in each group had organs from Table 5. Organs were not weighed for any female that failed to deliver a litter. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired reproductive organs were weighed separately; all other paired organs were weighed together.

HISTOPATHOLOGY: Yes (see Table 4 and Table 5)
The tissues listed in Table 4 were preserved for the first set of 5 P0 animals that survived until termination of dosing (Week 5 for males; LD 14 for females) as well as all females that failed to deliver a litter (GD 25). Slides of the indicated tissues were prepared and examined microscopically for all animals.

The tissues listed in Table 5 were preserved for all remaining P0 animals that survived until termin ation of dosing (Week 5 for males; LD 14 for females). Slides of the
indicated tissues were prepared and examined microscopically for all animals.

During the microscopic examination of the testes, special emphasis was placed on the stage of spermatogenesis and the interstitial testicular cell structure. Any cell- or stage-specificity of testicular findings was noted. During the microscopic examination of the ovary, qualitative depletion of the primordial follicle population was noted when present.

Eyes and testes were placed in Modified Davidson’s solution initially and then retained in 10% neutral buffered formalin (NBF). Lungs were infused with 10% NBF prior to their immersion into a larger volume of the same fixative. All other tissues were preserved in 10% NBF. After fixation, the tissues and organs from all animals were routinely processed, embedded in paraffin, sectioned at approximately 5microns, mounted on glass slides, stained with hematoxylin and eosin. All animals in Groups 1 and 4 were examined by light microscopy including animals in the intermediate dose groups where target organs were identified, animals that died prior to scheduled necropsy and females that failed to litter (GD 25).

Organ Weights:
The first five surviving adult males and lactating females that delivered a litter in each group had organs listed in Table 4. weighed and the second set of up to five surviving adult males and lactating females in each group had organs from Table 5. weighed. Organs were not weighed for any female that failed to deliver a litter. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired reproductive organs were weighed separately; all other paired organs were weighed together.

Postmortem examinations (offspring):

Pups were euthanized using an intraperitoneal injection of sodium pentobarbital. Macroscopic post-mortem examinations (external only) were performed on all surviving F1 pups on PND 13. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Unusual observations, including gross abnormalities were noted and then the carcasses were discarded.

F1pups that died during the study were given a macroscopic postmortem examination. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Unusual observations and the absence of milk in the stomach (up to PND 2), including gross abnormalities were noted. All carcasses were discarded following examination.

F1 pups selected for thyroid hormone blood collections were euthanized on PND 4 and discarded without further examination. The thyroid gland from one randomly selected male and female F1pup per litter was removed, fixed, weighed and stored in 10% neutral buffered formalin and processed for histopathology.

Statistics:

Please see ''Any other information on materials and methods incl. tables'' for information on Statistics.

Reproductive indices:

1) Female Mating Index (%):
(Number of females with confirmed mating (sperm and/or vaginal plug) + Number of pregnant females without evidence of mating (no sperm or vaginal plug) / (Number of females placed with males)) x 100

2) Female Fertility Index (%): ((Number pregnant) / (Number copulated)) x 100

3) Female Gestation Index (%): ((Number of females with live-born) / (Number of females with confirmed pregnancy)) x 100

4) Male Mating Index (%):
((Number of males with confirmed mating with a female or pregnancy for females without evidence of mating) / (Number of males placed with females)) x 100

5) Male Fertility Index (%):
((Number of males mating and impregnating a female + Number of males with a pregnant female without evidence of mating) / (Number of males with confirmed mating + number of males with a pregnant female without evidence of mating)) x 100.

Offspring viability indices:

1) Sex Ratio (% males): ((Number of male pups) / (number of total pups)) x 100

2) Viability Index (%): ((Number of pups alive on the specified day) / (Total number of live pups on Day 1)) x 100

3) Live Birth Index (%): ((Total number of live pups on Day 1) / (total number of pups born)) x 100

4) Post Implantation Survival (%): ((Number of live pups delivered) / (number of implantations)) x 100

5) Pre-Implantation Loss (%): ((Number of corpora lutea – Number of implantation sites) / (Number of corpora lutea)) x 100

6) Post-Implantation Loss (%): ((Number of implantation sites – Total number of live pups on Day 1) / (Number of implantation sites)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item-related reductions in the body weights and body weight changes at ≤200 mg/ Kg/day p-Cymene.

All animals during Pre-cohabitation (males and females), Cohabitation (males and females), Post-mating (males only) and Lactation (females only) with the exception of Animal Number 4572 either “Within Normal Limits”or had observations that were common to this strain of rodent in laboratory conditions. During the Gestation period, Animal Number 4572 was observed as being in poor condition and was sacrificed on presumed GD 24 for welfare reasons.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Eighteen females across all groups, including controls, were euthanized early. Seventeen of these females (1/10 control, 1/10 at 50 mg/Kg/day, 6/10 at 100 mg/Kg/day, and 9/10 at 200 mg/Kg/day) were euthanized on GD 25 due to failure to become pregnant. At the 200 mg/Kg/day dose level, females that failed to become pregnant was likely the result of germ cell degeneration/depletion and/or sperm retention in the testes of males in the 200 mg/Kg/day group. A minimal degree of sperm retention was present in some males at 100 mg/Kg/day which may have contributed to the reduced incidence of pregnancy in females at 100 mg/Kg/day.

One female at 200 mg/Kg/day (Female No. 4572) was euthanized for welfare reasons on GD 24; this female was not pregnant. Microscopic findings in this female were present in the liver, adrenal, and kidney and were considered the source of morbidity. Moderate, diffuse, micro/macrovesicular hepatocellular vacuolation was present in the liver, along with similar vacuolation in the cortex of the adrenal glands. In the kidney, bilateral tubular dilation and vacuolation (slight) were present in the cortex and bilateral tubular necrosis with degeneration/regeneration (slight) and neutrophilic inflammation were present in the papilla. No changes were present in the urinary bladder. The mechanism/cause of these findings in this one female were not determined by light microscopy. Although these findings were isolated to this one female, a test item effect could not be excluded, given that this was a high dose animal. Additional findings in this animal were considered stress-related and included lymphoid depletion (decreased cellularity) or necrosis/apoptosis in lymphoid tissues (thymus, mesenteric lymph node, splenic white pulp, Peyer’s patch/gut-associated lymphoid tissue) and atrophy of the splenic red pulp.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight changes in the 50, 100 and 200 mg/Kg/day p-Cymene male and female groups were comparable with control group values during Pre-cohabitation (males and females), Cohabitation (males and females), and Post-mating (males only) phases. The 200 mg/Kg/day p-Cymene female group body weights and body weight changes could not be evaluated during the gestation and lactation phases because none of the females were pregnant.

Body weights and body weight changes during the gestation and lactation phases in the 50 and 100 mg/Kg/day p-Cymene female groups were comparable with control group values.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption in the 50, 100 and 200 mg/Kg/day p-Cymene male and female groups were comparable with control group values during Pre-cohabitation (males and females), Cohabitation (males and females), and Post-mating (males only) phases. The 200 mg/Kg/day p-Cymene female group food consumption could not be evaluated during the gestation and lactation phases due to none of the females being pregnant. Food consumption during the gestation and lactation phases in the 50 and 100 mg/Kg/day p-Cymene female groups were comparable with control group values.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no p-cymene-related hematology changes at ≤ 100 mg/Kg/day.

p-Cymene-related hematology changes at 200 mg/Kg/day included increases in reticulocytes (1.30 × control; males only) with corresponding increases in red cell distribution width (1.07 × control; males only). These changes were considered non-adverse due to their relatively small magnitude.

There were no p-cymene-related coagulation changes at any dose. All differences between control and treated mean values, including those that were statistically significant, were not considered related top-Cymene as there was general overlap between individual control and treated values, and a lack of concordance between PT and APTT.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no p-cymene-related clinical chemistry changes at 50 mg/Kg/day. p-Cymene-related clinical chemistry changes at ≥ 100 mg/Kg/day included: decreases in triglycerides (0.50 to 0.52 × control; males only); increases in alkaline phosphatase activity (1.79 × control in females at 100 mg/Kg/day and 1.45 × control in males at 200 mg/Kg/day); and decreases in albumin (0.91 × control in females at 100 mg/Kg/day only). At 200 mg/Kg/day, increases in blood urea nitrogen (1.50× control; males only) were considered p-cymene related. All of these changes were considered non-adverse due to their relatively small magnitude.

All other differences between control and treated mean values, including those that were statistically significant, were considered to be unrelated to p-Cymene exposure as they lacked a dose relationship and/or there was general overlap between individual control and treated values.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional Observational Battery:
There were test item-related reductions in the 200 mg/Kg/day p-Cymene male group. The week 5 hindlimb grip strength was significantly reduced in the 200 mg/Kg/day p-Cymene male group. The LD 8 fore limb and the hindlimb grip strength in the 50 and 100 mg/Kg/day p-Cymene female groups and the Week 5 male fore limb grip strength in the 50, 100 and 200 mg/Kg/day p-Cymene were comparable to control group values.

Motor Activity:
There were no p-Cymene-related effects on male (Week 5) or female (LD 8) horizontal or vertical motor activity. All males in the 0, 50, 100 and 200 mg/Kg/day p-Cymene groups and all females in the 0, 50 and 100 mg/Kg/day p-Cymene groups showed habituation.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

p-Cymene-related microscopic findings were present in the reproductive tract of P0 males at ≥100 mg/Kg/day and in the liver of P0 males and/or females at 50 and 200 mg/Kg/day.

Testes and Epididymides:
Varying degrees of germ cell degeneration/depletion, depletion, and/or sperm retention were present in the testis at 200 mg/Kg/day. In some animals, this bilateral change consisted of multifocal drop out of round spermatids in early stage tubules (stages I to VIII), with germ cell degeneration, multinucleated giant cells, and vacuolation; general to multifocal depletion of elongated/mature spermatids in early stage tubules; germ cell drop out and disorganization in late stage tubules (stages X to XIII); bizarre mitotic figures in stage XIV tubules. In some animals, the findings consisted of multifocal depletion of round spermatids or depletion/degeneration of elongated spermatids in early stage tubules and occasional vacuolation. Most males also had sperm retention (retention of sperm in late stage tubules beyond stage VIII, stage at which sperm are released) along the apical surface of the tubules. In concert with the testicular findings, varying
degrees of luminal cell debris and reduced sperm with or without cribriform change were present bilaterally in the epididymis at 200 mg/Kg/day.

At 100 mg/Kg/day, some males had marginal sperm retention bilaterally in the testis with 2 of these males having decreased sperm in the epididymis with or without cribriform change. Unilateral or bilateral seminiferous tubular atrophy, with or without luminal cell debris and reduced sperm in the epididymis, was observed in 1 male (Animal No. 2028) at 50 mg/Kg/day and 1 male (Animal No. 3050) at 100 mg/Kg/day and was considered an incidental background finding and unrelated to findings attributed to p-Cymene. Isolated incidences of seminiferous tubular atrophy in rodents are not uncommon.

Liver:
Hepatocellular hypertrophy was minimally present in 2/5 males and 1/10 females at 200 mg/Kg/day and in 1/6 females at 50 mg/Kg/day. The distribution pattern of hepatocellular hypertrophy was diffuse at 200 mg/Kg/day and centrilobular at 50 mg/Kg/day. There were no degenerative changes associated with this finding. Focal hepatocellular necrosis and inflammatory cell infiltrates, observed sporadically or at a similar incidence in controls, were considered incidental background findings unrelated to hepatocellular hypertrophy and were not considered test article-related.

Kidney:
There was a marginal increase in incidence and severity of hyaline droplet accumulation in males at 200 mg/Kg/day compared to control males. Given the degree of increase was marginal, this change was likely incidental.

Minimal tubular epithelial vacuolation was present in the renal medulla in 2/5 males at 200 mg/Kg/day. The relationship of the medullary epithelial vacuolation to the test item was unclear.

All other microscopic findings occurred sporadically or at similar incidence and severity in control and p-Cymene-treated groups and were considered incidental background findings. Evaluation of primordial follicles in the ovary sections was limited because few primordial follicles were present in the sections for either controls or treated animals. Based on the tissues available for evaluation, there was no apparent differences between the controls and the 200 mg/Kg/day females.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Analysis:
At study termination, T4 values in P0 males were lower (0.44 to 0.63 × control) at 100 and 200 mg/Kg/day.Also, most or all of the individual animal TSH values were below the level of detection in P0 males at all doses and in P0 females at 50 and 100 mg/Kg/day. As there were no thyroid weight changes and/or microscopic findings in the thyroid glands in these animals the significance of these changes was considered unclear and likely not the result of test article administration.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

There were treatment-related alterations of Estrous Cyclicity at 200 mg/Kg/day. The number of animals with all regular cycles (cycles that have 4 day, 4/5 day and 5 day durations) during pre-cohabitation were reduced in the 100 and 200 mg/Kg/day dose groups but was considered adverse in the 200 mg/Kg/day dose group due to the magnitude. The number of animals with at least one irregular cycle (cycles < 4 days or > 5days duration) during the pre-cohabitation period were increased in the 200 mg/Kg/day dose group. The number of cycles and the cycle duration of the 50, 100 and 200 mg/Kg/day groups were comparable with the control values.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Lower mean testes, epididymides, and levator ani-bulbocavernosus muscle weights were present in P0 males at 200 mg/kg/day compared to controls. In the testes and epididymides, these weight differences, respectively, correlated microscopically with germ cell degeneration/depletion and decreased sperm, respectively.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

There were treatment-related reductions in fertility at ≥ 100 mg/Kg/day. The number of pregnant females was significantly reduced (4 and 0 females pregnant and the male and female fertility indices of 40% and 0%) in the 100 and 200 mg/Kg/day dose groups. The number of pregnant females were 9, 9, 4 and 0 in the 0, 50, 100 and 200 mg/Kg/day groups, respectively. The male and female fertility indices were 90%, 90%, 40% and 0% in the 0, 50, 100 and 200 mg/Kg/day groups, respectively.

The number of females paired with males were 10, 10, 10 and 10 in the 0, 50, 100 and 200 mg/Kg/day groups, respectively. The number of females that mated with males were 10, 10, 10 and 9 in the 0, 50, 100 and 200 mg/Kg/day groups, respectively. The number of females that had a defined GD 0 were 10, 10, 9 and 8 in the 0, 50, 100 and 200 mg/Kg/day groups, respectively. The mean number of days to mating were 2.5, 2.4, 3.2 and 2.6, respectively, was considered within normal limits. These mating values were comparable with the concurrent control values and were within normal historical data. Mating and fertility data of the 50 mg/Kg/day dose group were comparable with the concurrent control values.

Details on results (P0)

Analytical Chemistry:
The homogeneity results and concentration results for p-Cymenedose formulations of all groups analyzed during the course of the study met the study plan specified acceptance criteria. No test item was detected in the control samples. Therefore, all dose formulations used for dosing in this study met the acceptance criteria required by the study plan.

Haematological Findings:
p-Cymene-related hematology changes at 200 mg/Kg/day included increases in reticulocytes (1.30 × control; males only) with corresponding increases in red cell distribution width (1.07 × control; males only). These changes were considered non-adverse due to their relatively small magnitude.

Clinical Chemistry Findings:
There were no p-cymene-related clinical chemistry changes at 50 mg/Kg/day. p-Cymene-related clinical chemistry changes at ≥ 100 mg/Kg/day included: decreases in triglycerides (0.50 to 0.52 × control; males only); increases in alkaline phosphatase activity (1.79 × control in females at 100 mg/Kg/day and 1.45 × control in males at 200 mg/Kg/day); and decreases in albumin (0.91 × control in females at 100 mg/Kg/day only). At 200 mg/Kg/day, increases in blood urea nitrogen (1.50× control; males only) were considered p-cymene related. All of these changes were considered non-adverse due to their relatively small magnitude.

Functional Observational Battery:
There were test item-related reductions in the 200 mg/Kg/day p-Cymene male group. The Week 5 hindlimb grip strength was significantly reduced in the 200 mg/Kg/day p-Cymene male group.

Organ weight findings:
p-Cymene-related organ weight differences were present in the testes, epididymides, and levator anibulbocavernosus muscle in adult P0 males and in the liver in adult P0 males and females. Possible test material-related differences were present in the seminal vesicles and prostate in adult P0 males.

Gross pathology findings:
p-Cymene-related macroscopic findings were limited to adult (P0) males at 200 mg/Kg/day. Two males had unilateral (Male No. 4068) or bilateral (Male No. 4065) small testis. These findings correlated with microscopic findings of germ cell degeneration/depletion. One of these males (Male No. 4065) also had a small prostate; there was no correlating microscopic finding. One additional male (Male No. 4069) at 200 mg/Kg/day had a small levator ani-bulbocavernosus muscle complex; this
tissue was not examined microscopically.

Histopathology findings (non-neoplastic):
p-Cymene-related microscopic findings were present in the reproductive tract of P0 males at ≥100 mg/Kg/day and in the liver of P0 males and/or females at 50 and 200 mg/Kg/day.

Reproductive Function/Performance:
There were treatment-related alterations of Estrous Cyclicity at 200 mg/Kg/day. The number of animals with all regular cycles (cycles that have 4 day, 4/5 day and 5 day durations) during pre-cohabitation were reduced in the 100 and 200 mg/Kg/day dose groups but was considered adverse in the 200 mg/Kg/day dose group due to the magnitude. The number of animals with at least one irregular cycle (cycles < 4 days or > 5days duration) during the pre-cohabitation period were increased in the 200 mg/Kg/day dose group. The number of cycles and the cycle duration of the 50, 100 and 200 mg/Kg/day groups were comparable with the control values.

Lower mean testes, epididymides, and levator ani-bulbocavernosus muscle weights were present in P0 males at 200 mg/kg/day compared to controls. In the testes and epididymides, these weight differences, respectively, correlated microscopically with germ cell degeneration/depletion and decreased sperm, respectively.

There were treatment-related reductions in fertility at ≥ 100 mg/Kg/day. The number of pregnant females was significantly reduced (4 and 0 females pregnant and the male and female fertility indices of 40% and 0%) in the 100 and 200 mg/Kg/day dose groups. The number of pregnant females were 9, 9, 4 and 0 in the 0, 50, 100 and 200 mg/Kg/day groups, respectively. The male and female fertility indices were 90%, 90%, 40% and 0% in the 0, 50, 100 and 200 mg/Kg/day groups, respectively.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Systemic Toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Systemic Toxicity

3

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Reproductive Toxicity

4

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Reproductive Toxicity

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

cauda epididymis

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were 9, 9 and 4 litters that appeared normal for most of the observation period in the 0, 50 and 100 mg/Kg/day dose groups, respectively.

Minor observations, not considered treatment related, included: thin, cold to touch, partially absent appendages, little or no milk in stomach, swollen, twisted, encrustation, ulceration, dark color, pallor. These findings were not considered test material-related because they occurred only in the control group, occurred in the control group in similar incidence, did not occur in a treatment-related manner, was of short duration and/or was considered to be a common finding of young pups in a laboratory situation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There were treatment-related reductions in the mortality (decreased live birth index and the post-implantation survival index) at 100 mg/Kg/day.

There were no pregnant animals in the 200 mg/Kg/day dose group.

The live birth index was significantly reduced (94.3%) in the 100 mg/Kg/day dose group. The post-implantation survival index was reduced in the 100 mg/Kg/day dose group (87.3% compared with 95% in the concurrent control group). The number of litters with less than 100% viability was decreased in the 100 mg/Kg/day dose group with only 1 of 4 litters having 100% viability versus 9 of 9 in the concurrent control group.

The viability indices on PND 4, 7 and 13 were comparable with concurrent control values in the 100 mg/Kg/day dose group, respectively.

All offspring survival index values in the 50 mg/Kg/day dose group were comparable with the concurrent control values.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean Pup Weights on PND 1 were reduced in a treatment-related manner in the 100 mg/Kg/day dose group. The mean litter body weight was reduced in the 100 mg/Kg/day dose group on PND 1.

On the remaining intervals (PND 4, 7, 11 and 13), the mean litter body weights in the 50 and 100 mg/Kg/day dose groups were comparable with the control group values.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The mean sex ratio was comparable among groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related organ weight differences in thyroid/parathyroid glands in the F1pups.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no microscopic findings in the thyroid/parathyroid glands in the F1 pups.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone:
There were no differences from controls for thyroxine (T4) or thyroid stimulating hormone (TSH) on PND 4 in F1 female pups (Groups 1-3). On PND 13, there were no differences from control for T4 levelsin F1 male pups. Many of the individual animal TSH values were below the level of detection in males at 100 mg/Kg/day and females at 50 and 100 mg/Kg/day. As there were no thyroid weight changes and/or microscopic findings in the thyroid glands in these animals the significance of these changes was considered unclear and likely not the result of test article administration.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Clinical Signs:
There were 9, 9 and 4 litters that appeared normal for most of the observation period in the 0, 50 and 100 mg/Kg/day dose groups, respectively.

Minor observations, not considered treatment related, included: thin, cold to touch, partially absent appendages, little or no milk in stomach, swollen, twisted, encrustation, ulceration, dark color, pallor. These findings were not considered test material-related because they occurred only in the control group, occurred in the control group in similar incidence, did not occur in a treatment-related manner, was of short duration and/or was considered to be a common finding of young pups in a laboratory situation.

Mortality:
There were treatment-related reductions in the mortality (decreased live birth index and the post-implantation survival index) at 100 mg/Kg/day.

There were no pregnant animals in the 200 mg/Kg/day dose group.

The live birth index was significantly reduced (94.3%) in the 100 mg/kg/day dose group. The post-implantation survival index was reduced in the 100 mg/kg/day dose group (87.3% compared with 95% in the concurrent control group). The number of litters with less than 100% viability was decreased in the 100 mg/kg/day dose group with only 1 of 4 litters having 100% viability versus 9 of 9 in the concurrent control group.

The viability indices on PND 4, 7 and 13 were comparable with concurrent control values in the 100 mg/Kg/day dose group, respectively.

All offspring survival index values in the 50 mg/Kg/day dose group were comparable with the concurrent control values.

Body Weight:
The mean Pup Weights on PND 1 were reduced in a treatment-related manner in the 100 mg/Kg/day dose group. The mean litter body weight was reduced in the 100 mg/Kg/day dose group on PND 1.

On the remaining intervals (PND 4, 7, 11 and 13), the mean litter body weights in the 50 and 100 mg/Kg/day dose groups were comparable with the control group values.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental Toxicity

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 6. Hematology Results – group mean values
Group (Sex)		RETIC x10^9/L	RDW %	PT Seconds
Control	Mean	163.4	12.5	18
	SD	9.51	0.19	0.76
	N	4	4	5
50 mg/Kg/day (Males)	Mean	166.3	12.7	18.7
	SD	20.02	0.49	0.54
	N	5	5	5
100 mg/Kg/day (Males)	Mean	151.8	12.3	19.5**
	SD	15.96	0.36	0.77
	N	5	5	5
200 mg/Kg/day (Males)	Mean	211.7**	13.4*	19.6**
	SD	36.29	0.80	0.40
	N	5	5	5

* p<0.05

** p<0.01

 

Table 7. Clinical Chemistry Results – group mean values
Group (Sex)		ALKP U/L	BUN mg/dL	CREAT mg/dL	TRIG mg/dL	Na^+ mEq/L	Cl^- mEq/L	PHOS mg/dL
Control	Mean	160	12	0.3	86	143	103	8.5
	SD	23.5	0.0	0.05	14.4	1.4	1.1	0.41
	N	5	4	5	5	5	5	5
50 mg/Kg/day (Males)	Mean	166	13	0.3	69	143	102*	7.9
	SD	43.5	0.9	0.04	38.1	0.8	1.2	0.50
	N	5	5	5	5	5	5	5
100 mg/Kg/day (Males)	Mean	184	14	0.3	45*	143	102*	7.8*
	SD	22.8	1.3	0.0	9.1	0.5	0.9	0.11
	N	5	5	5	5	5	5	5
200 mg/Kg/day (Males)	Mean	232*	18**	0.3*	43**	141*	102*	8.5
	SD	61.4	1.8	0.05	18.1	0.5	0.8	0.43
	N	5	5	5	5	5	5	5

* p<0.05

** p<0.01

 

Table 8. Clinical Chemistry Results – group mean values
Group (Sex)		ALT U/L	ALKP U/L	CHOL mg/dL	ALB g/dL
Control	Mean	183	151	120	3.5
	SD	39.0	19.8	30.8	0.29
	N	5	5	5	5
50 mg/Kg/day (Females)	Mean	124**	210	95	3.5
	SD	25.9	70.9	12.0	0.15
	N	5	5	5	5
100 mg/Kg/day (Females)	Mean	93**	270*	85*	3.2*
	SD	14.0	119.1	5.4	0.21
	N	4	4	4	4

* p<0.05

** p<0.01

 

Table 9. Functional assessment – Group summary of assessments (males – week 5)
Dose Group		Vehicle	p-Cymene
Dose (mg/Kg/day)		1 (Control)	2 (50)	3 (100)	4 (200)
Forelimb Grip Strength (g)	Mean	1401.7	1109.3	1143.7	984.4
	SD	346.3	413.1	261.7	231.9
	N	5	5	5	5
Hindlimb Grip Strength (g)	Mean	837.7	680.5	628.2	541.6*
	SD	284.5	54.2	152.0	227.7
	N	5	5	5	5

* p<0.05

 

Table 10. p-Cymene-related organ weight changes in male reproductive organs (% difference relative to controls) in adult P0 male rats at the end of dosing.
Group (sex)	2 (M)	3 (M)	4 (M)
Dose (mg/Kg/day)	50	100	200
Testes^b
Absolute weight (%)	-	-	-14^a
vs. body weight (%)	-	-	-8
vs. brain weight (%)	-	-	-12
Epididymides^b
Absolute weight (%)	-	-	-14^a
vs. body weight (%)	-	-	-8
vs. brain weight (%)	-	-	-14^a
Levator Ani-Bulbocavernosus Muscle^b
Absolute weight (%)	-	-	-14
vs. body weight (%)	-	-	-9
vs. brain weight (%)	-	-	-15
Seminal Vesicles/ Coagulating glands
Absolute weight (%)	-19	-23	-22
vs. body weight (%)	-20	-22	-14
vs. brain weight (%)	-16	-23	-18
Prostate
Absolute weight (%)	-26	2	-24^a
vs. body weight (%)	-27^a	5	-16
vs. brain weight (%)	-23	3	-20

^a Statistically significant difference between mean values for test item-treated and control groups.

^b Values in table represent those calculated from data combining toxicity and reproductive subsets of

animals.

- = not test item-related.

 

Table 11. p-Cymene-related organ weight changes in liver (% difference relative to controls) in adult P0 male rats at the end of dosing and adult P0 female rats at the end of lactation
Sex	Male			Female
Dose (mg/Kg/day)	50	100	200	50	100	200
Liver
Absolute weight (%)	8	6	27^a	16	26^a	NT
vs. body weight (%)	6	8^a	41^a	14	22^a	NT
vs. brain weight (%)	13	6	35^a	14	22	NT

^a Statistically significant difference between mean values for test item-treated and control groups.

NT = not taken; no organ weights were taken for females at 200 mg/Kg/day as all were euthanized prior to scheduled termination due to non-pregnancy.

 

Table 12. p-Cymene-related findings in the testis and epididymis in adult P0 male rats at the end of dosing
Group (Sex)	1 (M)	2 (M)	3 (M)	4 (M)
Dose (mg/Kg/day)	0	50	100	200
Testis^a
Degeneration/Depletion, Germ Cell
Minimal	-	-	-	1
Slight	-	-	-	5
Moderate	-	-	-	1
Total	0	0	0	7
Depletion, Germ Cell
Minimal	-	-	-	2
Total	0	0	0	2
Retention, Spermatid
Minimal	-	-	7	2
Slight	-	-	-	7
Total	0	0	7	9
Epididymides^a
Sperm, Reduced, Luminal
Minimal	-	-	1	-
Slight	-	-	1	4
Moderate	-	-	-	5
Marked	-	-	-	1
Total	0	0	2	10
Cribriform Change
Minimal	-	-	-	2
Slight	-	-	1	3
Total	0	0	1	5
Cell Debris, Luminal
Minimal	-	-	-	2
Slight	-	-	-	3
Moderate	-	-	-	4
Total	0	0	0	9
Number of Animals Examined	10	10	10	10

^aAll changes were bilateral.

- = Finding not present.

 

Table 13. p-Cymene-related findings in the liver in adult P0 male rats at the end of dosing and adult P0 female rats at the end of dosing/lactation.
Group (Sex)	1 (M)	2 (M)	3 (M)	4 (M)	1 (F^a)	2 (F^a)	3 (F^a)	4 (F^a)
Dose (mg/Kg/day)	0	50	100	200	0	50	100	200
Hypertrophy, Hepatocellular
Minimal	-	-	-	2	-	1	-	1
Total	0	0	0	2	0	1	0	1
Number of Animals Examined	5	5	5	5	6	6	10	10

- = Finding not present.

^a Females include all animals in which liver was examined including those euthanized during gestation period that were not pregnant.

 

Table 14. Histopathology: p-Cymene-related findings in kidneys in adult P0 Male and Female Rats.
	Group/Sex	1 (M)	2 (M)	3 (M)	4 (M)	1 (F)	2 (F)	3 (F)	4 (F)
	Dose (mg/Kg/day)	0	50	100	200	0	50	100	200
	No. of animals	10	10	10	10	9	9	4	0
Tissue/Organ and Findings
Kidneys	No. examined	5	5	5	5	5	5	4	0
Dilatation, Tubular	Slight	1	0	0	0	0	1	0	0
	Total	1	0	0	0	0	1	0	0
Vacuolation, Tubular Epithelium	Minimal	0	0	0	2	0	0	0	0
	Total	0	0	0	2	0	0	0	0
Accumulation, Hyaline Droplets	Minimal	1	1	0	2	0	0	0	0
	Slight	0	0	0	1	0	0	0	0
	Total	1	1	0	3	0	0	0	0
Basophilia, Tubular	Minimal	0	1	1	1	0	0	0	0
	Total	0	1	1	1	0	0	0	0

Table 15. Estrous cycle evaluations – group mean values
Group (sex)	Number of Animals	Regular Cycles				Irregular Cycle^a	Extended Estrus^b	Acyclic^c
		4 Day	4/5 Day	5 Day	Total
Control (Female)	10	6	0	0	6	4	0	0
		60%	0%	0%	60%	40%	0%	0%
50 mg/Kg/day (Female)	10	7	0	0	7	3	0	0
		70%	0%	0%	70%	30%	0%	0%
100 mg/Kg/day (Female)	10	4	3	0	7	3	0	0
		40%	30%	0%	70%	30%	0%	0%
200 mg/Kg/day (Female)	10	1	3	0	4	6	0	0
		10%	30%	0%	40%	60%	0%	0%

a – At least one cycle of < 4 or > 5 days

b – At least four consecutive days of estrus

c – At least ten days without estrus

 

Table 16. Mating and fertility – group mean values
	Dose Group	Control	50 mg/Kg/day	100 mg/Kg/day	200 mg/Kg//day
Females paired with males	N	10	10	10	10
Total number mated	N	10	10	10	9
female mating index	%	100	100	100	90
Total number pregnant	N	9	9	4	0
female fertility index	%	90	90	40	0
Males placed with females	N	10	10	10	10
Total number mated	N	10	10	10	9
male mating index	%	100	100	100	90
with females pregnant	N	9	9	4	0
male fertility index	%	90	90	40	0
Females with defined
Day0 of Gestation	N	10	10	9	8
Number of days to Mating	Mean	2.5	2.4	3.2	2.6
	SD	0.97	0.97	0.97	0.92
Day 1 to 4	N	10	10	8	8
	%	100	100	88.9	100
Day 5 to 8	N	0	0	1	0
	%	0	0	11.1	0
Day 9 to 14	N	0	0	0	0
	%	0	0	0	0

 

Table 17. Offspring survival indices - group mean values
Group (sex)		Post Implantation Survival Index (%)	Live Birth Index (%)	Viability Index (%) Day 4	Viability Index (%) Day 7^a	Viability Index (%) Day 13^a
Statistics Test		Wi	CA	Ch	+	+
Control (Female)	Mean	95.0	100.0	100	86.7	84.5
	SD	6.55	0.00	0.00	1.24	3.27
	N	9	9	9	9	9
	N<100%	4	0	0	9	9
50 mg/Kg/day (Female)	Mean	97.7	97.7	100.0	84.3	84.3
	SD	4.65	4.77	0.00	4.28	4.28
	N	9	9	9	9	9
	N<100%	2	2	0	9	9
100 mg/Kg/day (Female)	Mean	87.3	94.3	98.3	82.5	82.5
	SD	14.50	4.17	3.33	4.33	4.33
	N	4	4	4	4	4
	N<100%	3	3**	1	4	4

The following data were used for the statistics tests, animal indices for post implantation survival index and animal indices dichotomized to 1 when 100% and 0 otherwise for live birth and viability indices.

A + indicates statistics (Fisher's exact tests) were only possible on additional pairwise comparisons.

a – Offspring removed for hormone analysis are not excluded from calculation of the viability index.

Table 18. Litter size - group mean values
Group (sex)		Gestation Length	Corpora Lutea	Implantations	Pre-Implantation Loss (%)	Total @	Live on Day
							1	4	7	11	13
Statistics Test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
Control (Female)	Mean	21.8	16.3	16.0	1.8	15.1	15.1	15.1	13.1	12.9	12.8
	SD	0.44	2.40	2.06	3.64	1.45	1.45	1.45	1.45	1.36	1.39
	N	9	9	9	9	9	9	9	9	9	9
50 mg/Kg/day (Female)	Mean	21.7	14.9	14.0	5.9	13.8	13.4	13.4	11.4	11.4	11.4
	SD	0.50	3.06	3.08	6.81	2.99	2.96	2.96	2.96	2.96	2.96
	N	9	9	9	9	9	9	9	9	9	9
100 mg/Kg/day (Female)	Mean	21.3	17.3	16.0	7.1	14.0	13.3	13.0	11.0	11.0	11.0
	SD	0.58	1.89	1.41	1.94	2.71	2.87	2.71	2.71	2.71	2.71
	N	3	4	4	4	4	4	4	4	4	4

@ - Includes offspring that died prior to the designated Day 1 of age

Table 19. Body weight - group mean values (g) for offspring
Group (Sex)		Day of Age
		1	4	7	11	13
Statistics Test		Wi	Wi	Wi	Wi	Wi
Control (Male)	Mean	6.8	8.9	13.1	20.4	24.5
	SD	0.62	0.89	1.31	2.08	2.70
	N	9	9	9	9	9
50 mg/Kg/day (Male)	Mean	6.8	9.8	14.8	23.0	26.8
	SD	0.91	1.61	2.68	4.77	5.22
	N	9	9	9	9	9
100 mg/Kg/day (Male)	Mean	6.1	8.7	13.4	21.2	25.1
	SD	0.44	0.50	0.78	1.58	1.72
	N	4	4	4	4	4
Control (Female)	Mean	6.6	8.4	12.7	20.0	23.7
	SD	0.66	0.90	1.51	2.74	3.25
	N	9	9	9	9	9
50 mg/Kg/day (Female)	Mean	6.3	9.0	14.0	22.2	26.0
	SD	0.84	1.75	2.80	4.85	5.53
	N	9	9	9	9	9
100 mg/Kg/day (Female)	Mean	6.0	8.6	12.8	19.9	23.7
	SD	0.30	0.53	0.81	1.04	1.16
	N	4	4	3	3	3

Applicant's summary and conclusion

Conclusions:

Based on the results observed, the systemic toxicity NOAEL for P0 males was determined to be 50 mg/Kg/day, based on epididymal and testicular organ weights, testicular germ cell degeneration/depletion and/or sperm retention in the testes and epididymal luminal cell debris and reduced sperm and cribriform changes at 100 or 200 mg/Kg/day. Based on alterations in oestrous cyclicity and morbidity at 200 mg/Kg/day the, NOAEL for P0 females was considered to be 100 mg/Kg/day.
 
The NOAEL for F1 offspring is considered to be 50 mg/Kg/day, based on decreased pup live birth index, decreased post-implantation survival index, decreased pup viability, decreased pup weights and litter weights in the 100 mg/Kg/day dose group on PND 1.

Executive summary:

In a key combined repeated dose toxicity with reproduction/developmental toxicity screening study in the rat, the potential effects of repeated oral gavage administration of the test material (p-Cymene; CAS# 99-87-6) on male and female reproductive performance and systemic toxicity were evaluated in accordance with OECD Test Guideline 422.

 

The test material was administered to Sprague-Dawley CD^®rats (10 animals/sex/group) were via oral gavage once daily at 0 (corn oil), 50, 100 or 200 mg/Kg/day. P0 males were dosed during the pre-cohabitation, cohabitation and post-mating periods (approximately 35 days) and then euthanized and necropsied. P0 females were dosed during the pre-cohabitation and cohabitation periods and during gestation and lactation through lactation Day 13 for approximately 63 days and then euthanized and necropsied. Parameters evaluated during the study for the P0 animals were: viability, clinical observations, body weights, food consumption, estrous cycling, mating and fertility, parturition and littering, functional assessments including motor activity, clinical pathology (termination), hormone analysis, organ weights, macroscopic observations and microscopic pathology.

 

No test material-related mortalities were observed during the study in males or females at ≤100 mg/Kg/day. No adverse test material-related clinical observations were observed during the study in males at ≤ 200 mg/Kg/day and in females at ≤ 100 mg/Kg/day. In adult (P0) females (except animal no.4572), there were no macroscopic findings considered to be treatment-related. There were no test material-related reductions in the body weights, body weight changes and food consumption in either gender at ≤ 200 mg/Kg/day.

 

Week 5 hindlimb grip strength was significantly reduced in the 200 mg/Kg/day males. The LD 8 fore limb and the hindlimb grip strength in the 50 and 100 mg/Kg/day female groups and the Week 5 male fore limb grip strength in the 50, 100 and 200 mg/Kg/day dose groups were comparable to control group values.

There were no adverse treatment-related haematology, clinical chemistry or coagulation changes observed at ≤ 200 mg/Kg/day. There were no macro- and/or microscopic findings or organ weight changes in the reproductive tract of females at 50, 100, or 200 mg/Kg/day where examined.

 

Elevated liver weights were observed in males at 200 mg/Kg/day (and minimally at 50 and 100 mg/Kg/day) and in females at 50 and 100 mg/Kg/day (no organ weight data for 200 mg/Kg/day females).

 

Treatment-related reductions in male fertility and male fertility index were observed at ≥ 100 mg/Kg/day. There were test material-related testicular changes observed at 200 mg/Kg/day (lower organ weights, germ cell depletion/degeneration, depletion, and/or sperm retention), along with correlative changes in the epididymis that were considered adverse. These changes were considered the likely cause of the total absence of pregnancy among P0 females in the 200 mg/Kg/day dose group. At 100 mg/Kg/day, a marginal degree of sperm retention was observed and this may have contributed to the absence of pregnancy in 60% of P0 females at the mid-dose level.

 

Test material-related alterations of oestrous cyclicity, including a reduction of the number of animals with all regular cycles and an increase in the number of females with at least one irregular cycle, were observed in the 200 mg/Kg/day dose group females. There were treatment-related reductions in female fertility and female fertility index at ≥100 mg/Kg/day. The number of females that were pregnant were significantly reduced at ≥100 mg/Kg/day dose level.

 

No litters delivered at 200 mg/Kg/day (the highest dose tested) and treatment-related reductions in litter viability were observed at 100 mg/Kg/day, the highest dose at which litters were delivered. In addition, treatment-related alterations in pup mortality (decreased live birth index and the post-implantation survival index) were observed at 100 mg/Kg/day. Mean pup weights and mean litter weights were reduced in the 100 mg/Kg/day dose group on PND 1. At study termination, T4 values were reduced (0.44 to 0.63 of control) in F1 males only at 100 and 200 mg/Kg/day.

 

Based on the results observed, the systemic toxicity NOAEL for P0 males was determined to be 50 mg/Kg/day, based on epididymal and testicular organ weights, testicular germ cell degeneration/depletion and/or sperm retention in the testes and epididymal luminal cell debris and reduced sperm and cribriform changes at 100 or 200 mg/Kg/day. Based on alterations in oestrous cyclicity and morbidity at 200 mg/Kg/day the, NOAEL for P0 females was considered to be 100 mg/Kg/day.

 

The NOAEL for F1 offspring is considered to be 50 mg/Kg/day, based on decreased pup live birth index, decreased post-implantation survival index, decreased pup viability, decreased pup weights and litter weights in the 100 mg/Kg/day dose group on PND 1.