2,4-dimethyl-4-phenyltetrahydrofuran

Endpoint summary

Description of key information

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

- NOAEL for F0 male and female reproductive and systemic toxicity: 600 mg/kg bw/day, based on a lack of adverse findings noted during the premating period or on neurobehavior, motor activity, and pathology parameters and based on no effects on male or female reproductive indices at any dose level (OECD 422 - oral gavage, GLP, Rel.1)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 October 2020 to 29 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 422 without any deviation

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately 11 weeks old.
- Weight at study initiation: between 208 and 427 g at the initiation of dosing.
- Fasting period before study: no
- Housing: Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study and grouped up to 3 animals of the same sex until cohabitation and separated during designated procedures/activities. During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system. Water bottles were provided, if required.
- Acclimation period: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.
- Environmental enrichment: Animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental
contaminants. Results of the analysis are provided by the supplier. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are provided by the supplier. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2020-10-05 To: 2020-12-27

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was chosen because it is a potential route of exposure to humans and
to comply with regulatory requirements.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (target of 5°C, protected from light, and purged with nitrogen until use. The dosing formulations were stirred continuously during dosing.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly

VEHICLE
- Vehicle: Corn oil. The vehicle was dispensed weekly for administration to Group 1 control animals and preparation of the test substance formulations. For administration to Group 1 control animals, an adequate amount of the vehicle was dispensed into daily aliquot. The vehicle was stirred continuously during dosing.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Storage Conditions: refrigerated (target of 5°C), protected from light, and purged with nitrogen until use.
- Supplier: Charles River Laboratories

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analytical method:
Samples to be analyzed were transferred to the Charles River Ashland Analytical Chemistry
Department. Analyses described below were performed by a gas chromatographic method with flame ionization detection using a validated analytical procedure (Akalkotkar, 2020, 00810043):
- Analysis of concentration: all groups
- Analysis of homogeneity: Groups 2 and 4

- Concentration Analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration, with each individual sample concentration within ± 20% of the target concentration

- Homogeneity analysis:
Duplicate sets of samples (1.0 mL) from the first sampling time point were transferred to the
analytical laboratory. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration.
Test substance formulations have been previously shown to be homogenous over the range of concentrations used on this study for at least 8 days at a target temperature of 5°C (Akalkotkar,
2020, 00810043). Therefore, resuspension homogeneity of test substance formulations was not assessed on this study.

- Stability analysis:
Test substance formulations have been previously shown to be stable over the range of concentrations used on this study for at least 8 days at a target temperate of 5°C (Akalkotkar,
2020, 00810043). Therefore, stability of test substance formulations was not assessed on this
study.

Duration of treatment / exposure:

Males were dosed for 14 days prior to mating, throughout mating and continuing through mating for a minimum of 28 days.
Females were dosed for 14 days prior to mating, and continuing through mating, gestation, and lactation until 1 day prior to scheduled euthanasia (minimum of Study Days 0–38 or maximum of Study Days 0–60 based on breeding success). Females that failed to deliver were dosed through the day prior to euthanasia.
The F1 animals were not directly exposed to the test article at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test article in utero and while nursing.

Frequency of treatment:

All animals were dosed at approximately the same time each day using appropriately sized flexible, disposable plastic feeding tubes.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 -Vehicle

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 - test item

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 3 - test item

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

Group 4 - test item

No. of animals per sex per dose:

10 animals/sex/dose

RATIONALE FOR NUMBER OF ANIMALS:
The number of animals selected for this study was based on the OECD 422 Guideline for the Testing, which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group.
Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycle (e.g., EDDDE), during the pretest period, were excluded from the study; therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE:
The dosage levels were determined based on the results of a 14-day oral gavage toxicity study with rhubafuran in rats (Harrison, Draft, 00810044). In that study, test substance administration at 1000 mg/kg/day resulted in moribund euthanasia of 4 females and transient clinical observation of decreased activity. A subsequent reduction in dose to 800 mg/kg/day following a dosing holiday on Study Day 2 resulted in similar transient decreased activity observations which were not observed for any animal past Study Day 6. No test substance-related clinical observations were noted in any other dose group; however, increased liver weights were observed at ≥ 300 mg/kg/day for males and ≥ 600 mg/kg/day for females compared to the concurrent control group. Based on these results, a high dose of 600 mg/kg/day was selected for the current study as it was expected to demonstrate toxicity of the test substance without resulting in death or suffering. Lower doses were selected at appropriate intervals in or to assess dose response relationship.

SELECTION, ASSIGNMENT AND DISPOSITION OF ANIMALS
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.
To reduce variability among the F1 litters, 8 pups/litter of equal sex distribution, if possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. The remaining offspring were euthanized by an intraperitoneal injection of sodium pentobarbital (following thyroid hormone blood collection for pups used for blood collection) and discarded.

Observations and examinations performed and frequency:

MORTALITY/VIABILITY: Yes
- Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- The animals were removed from the cage, and a detailed clinical observation was performed
once daily throughout the study. During the dosing period, these observations were performed
prior to dosing. On dosing days, clinical observations were also recorded approximately 1 hour postdose. During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 10, 13 and 14. A fasted weight was recorded on the day of necropsy. Terminal body weightswere not collected from animals euthanized in extremis.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured twice weekly until cohabitation. Once evidence
of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, and 13.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY, CLINICAL CHEMISTRY AND THYROID HORMONE ANALYSIS: Yes
- Hematology, Coagulation and serum chemistry analysis were performed on all groups at Study Day 28 for males and Lactation Day 14 for females.
Animals were fasted overnight prior to blood collection. Blood samples for hematology and serum chemistry were collected from a jugular vein for males and from the retro-orbital sinus from animals anesthetized with isoflurane for females. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation. K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for serum chemistry were collected without anticoagulants.
- Parameters checked in table 7.5.1/1 were examined.


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- FOB assessments recorded for 5 animals/sex/group during the last week of dosing (Day 27; F0 males) or on Lactation Day 13 (F0 females). Testing was performed by the same trained technicians, when possible, who did not know the animal’s group assignment and was performed at approximately the same time each day. The FOB was performed in a sound-attenuated room equipped with a white noise generator. All animals were observed for the following parameters:
- Activity: Rearing/Arousal/Alertness/Posture/Body Carriage/Stereotypy/Appearance
- Autonomic: Exophthalmus/Lacrimation/Erected Fur/Palpebral Closure/Ptosis/Rearing/Defecation/Urination/Salivation/Pupil Response
- Excitability: Vocalizations/Startle Response/Handling Reactivity/Convulsions/Ease of removal
Neuromuscular: Gait/Mobility/Grip Strength/Body Tone/Tremor/Air Righting Reflex
- Physiological: Respiration/Body Temperature
- Sensorimotor: Pain/Tail Pinch Response/Touch Response/Tactile Reflex
- Locomotor activity was assessed for 5 animals/sex/group during the last week of dosing (Day 27; F0 males) or on Lactation Day 13 (F0 females). The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator, and black enclosures were used to decrease the potential for distraction. Data were collected in 5-minute epochs over a period of 60 minutes, and the data were reported in 10-minute subintervals. Total motor activity was defined as a combination of fine motor skills (i.e., grooming; interruption of 1 photobeam) and ambulatory motor activity (e.g., interruption of 2 or more consecutive photobeams).

IMMUNOLOGY: No

OTHER: Cf. IUCLID 7.8.1
- Estrous cycle
- Mating procedure
- Parturition observation and gestation length

Sacrifice and pathology:

Terminal procedures were presented below in Table 7.5.1/2.

GROSS PATHOLOGY: Yes
- Unscheduled Deaths: If necessary for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained. For females euthanized during lactation, the number of former implantation sites were recorded.
- Scheduled Euthanasia: All surviving animals were euthanized by carbon dioxide inhalation.
- Necropsy: Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. Postimplantation loss was calculated for each female by subtracting the number of pups born from the number of implantation sites observed. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide
solution for detection of early implantation loss.

ORGAN WEIGHTS: Yes
- The following organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals euthanized in extremis. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
- Organs Weighed at Necropsy: Adrenal glands/Brain/Epididymides/Heart/Kidneys/Liver/Ovaries with oviducts/Pituitary gland/Prostate gland/Seminal vesicle (with coagulating gland and fluid)/Spleen/Testes/Thymus gland/Thyroids with parathyroids (after fixation).
- Representative samples of the tissues identified in Table 7.5.1/3 were collected from all animals
and preserved in 10% neutral buffered formalin, unless otherwise indicated.

HISTOLOGY: Yes
- Tissues identified in Table 7.5.1/3 from 5 animals/sex in the control and high-dose groups and from all animals dying spontaneously, as well as gross lesions from all groups, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides. The liver was identified as a target tissue and was prepared for the remaining animals in the control and high-dose groups, and from all parental animals in the low- and mid-dose group.

HISTOPATHOLOGY: Yes
- Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues
identified in table 7.5.1/3 for microscopic examination were evaluated from 5 animals/sex in
the control and high-dose groups and from all animals euthanized in extremis. Gross lesions were examined from all groups. In addition, the liver was identified as a target tissue and was examined from the remaining animals in the control and high-dose group and from all parental animals in the low- and mid-dose group.

Optional endpoint(s):

Optional endpoints: No

Statistics:

Data collected during the predose period were not tabulated, summarized, or statistically analyzed, unless applicable to analyses in the proceeding sections. All statistical analyses were performed within the respective study phase, unless otherwise noted. Clinical and necropsy observations data were summarized but no inferential statistical analysis was performed.
Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion or by litter. Values may also be expressed as a percentage of pretreatment period or control values, or fold change of control values, when deemed appropriate. Calculated values on Provantis tables may not be reproducible from the individual values presented because all calculations were conducted using non-rounded values.

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Analyses were performed according to the matrix attached in the full report study attached (p.38 and 39) when possible, but will exclude any group with less than 3 observations.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 600 mg/kg/day group, clinical observations were noted for the Female No. 4508 prior to euthanasia including pallor skin during Lactation Days 1–5, red fur staining around the urogenital area and pale skin on numerous surfaces on Lactation Day 0, slight salivation on Lactation Day 1, and increased respiratory rate, thin body, cold to the touch, and red fur staining around the nose on Lactation Day 5.
These findings were consistent with bacterial sepsis (possibly secondary to endometritis), which was deemed the cause of debility in this animal and was likely associated with the clinically observed dystocia. This death was considered secondary to dystocia and not directly related to rhubafuran administration.

In the 600 mg/kg/day groups, clinical observations of wet fur around the mouth and/or muzzle were noted approximately 1 hour following dose administration for 4 males during Study Days 13–24 and for 3 females during Study Days 10–15, 4 females during Gestation Days 0–9, and for 7 surviving females during Lactation Days 6–13 compared to 0 instances in the control group. In addition, slight to moderate salivation was noted for 6 surviving females in the 600 mg/kg/day group approximately 1 hour following dose administration during Lactation Days 1–13 compared to 0 instances in the control group. Wet fur around the muzzle was also noted for 1 male in the 200 mg/kg/day group approximately 1 hour following dose administration on Study Day 24 and slight to moderate salivation and/or wet fur around the mouth were noted approximately 1 hour following dose administration for 2 females in the 200 mg/kg/day during Lactation Days 2–11.
These clinical observations were considered test substance-related but not adverse because they did not persist to the daily examinations and had no impact on any other parameter evaluated. Additionally, these observations may have been associated with unpleasant smell and/or taste of the test substance due to its olfactory potency. Other clinical observations were noted infrequently, similarly in the control group, and/or in a manner that was not dose-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the 600 mg/kg/day group, Female No. 4508 was euthanized in extremis on Lactation Day 5.
This female delivered 3 live pups and 3 dead pups, and then delivered 6 dead pups 2 days later, indicating signs of dystocia. These findings were consistent with bacterial sepsis (possibly secondary to endometritis), which was deemed the cause of debility in this animal and was likely associated with the clinically observed dystocia. This death was considered secondary to dystocia and not directly related to rhubafuran administration.

All other F0 males and females in the control, 100, 200, and 600 mg/kg/day groups survived to the scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight gains in the 100, 200, and 600 mg/kg/day group males were unaffected by test substance administration throughout the study. None of the differences from
the control group were statistically significant.

Mean body weights and body weight gains in the 100, 200, and 600 mg/kg/day group females were unaffected by test substance administration during the premating period, the gestation and the lactation periods. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day, in the 100, 200, and 600 mg/kg/day group males was unaffected by test substance administration throughout the study. No statistically significant differences were observed.
Mean food consumption, evaluated as g/animal/day, in the 100, 200, and 600 mg/kg/day group females was unaffected by test substance administration during the premating period, the gestation and the lactation periods. None of the differences from the control group were statistically significant.

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):

No test substance-related effects on hematology parameters were noted. Statistically significantly lower mean corpuscular hemoglobin was noted for males in the 200 and 600 mg/kg/day groups and lower mean corpuscular hemoglobin concentration was noted for males in the 100 and 600 mg/kg/day groups compared to the control group. These differences were only noted for 1 sex, were not noted in a dose-related manner, and/or were within the values of the Charles River Ashland historical control data (version 3.6), and therefore were not considered test substance-related.

Other differences from the control group values were not statistically significant, did not occur in a dose-related manner, were only observed in 1 sex and/or were of minimal magnitude.

No test substance-related effects on coagulation were noted. A statistically significantly lower mean prothrombin time was noted in the 600 mg/kg/day group females compared to the control group; however, this difference was only noted for 1 sex and therefore was attributed to biologic variation. Other differences from the control group values were not statistically significant and/or did not occur in a dose-related manner.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly higher mean total protein and globulin concentrations were noted in the 100 and 600 mg/kg/day group males in a non-dose-related manner. In addition, higher mean cholesterol concentrations were noted for males in all test substance-treated groups and for females at 200 and 600 mg/kg/day compared to the control group; differences were statistically significant for F0 females at 600 mg/kg/day only.
These effects were not considered test substance related as they were within the range of values observed in the historical control data (males) and/or there were no correlates observed in the clinical pathology or microscopic data.

Other differences from the control group were slight, not statistically significant, and/or only noted for 1 sex, and therefore were not considered test substance-related.

Endocrine findings:

no effects observed

Description (incidence and severity):

Mean T3, T4, and TSH concentration in the 100, 200, and 600 mg/kg/day group F0 males were unaffected by test substance administration. Differences from the control group were not statistically significant and/or not noted in a dose-related manner.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Neurobehavioral assessments were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group on Study Day 27 (males) or on Lactation Day 13 (females).

Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all concentrations when evaluated on Study Day 27 (males) or Lactation Day 13 (females). Values obtained from the 6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data. Differences from the control group were slight, not statistically significant, within the Charles River Ashland historical control data ranges and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher mean kidney weights were noted in the 600 mg/kg/day group with some individual weights being above the historical control range as well, but there was not a microscopic correlate for the difference. Higher mean liver weights were noted in the 200 mg/kg/day group males and in the 600 mg/kg/day group in both sexes. This correlated to hepatocellular hypertrophy microscopically.
These organ weight increases were considered adaptive and non-adverse.

No other test substance-related organ weight changes were noted.
There were other isolated organ weight values that were different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, other organ weight differences observed were considered incidental and unrelated to administration of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were noted.
The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in the control and treated animals, and therefore, were considered unrelated to administration of the test substance.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the liver. Hepatocellular hypertrophy was characterized by slightly enlarged hepatocytes with increased cytoplasm, particularly in centrilobular regions. This finding correlated with higher mean liver weights in the 200 mg/kg/day group males and in the 600 mg/kg/day group in both sexes.
Hepatocyte hypertrophy is commonly associated with microsomal enzyme induction secondary to exposure to certain xenobiotics and was therefore considered adaptive and non-adverse (Cattley and Popp, 2002).
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals, and therefore, were considered unrelated to administration of the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Cf. IUCLID section 7.8.1 for results linked to reproductive and developmental toxicity

Details on results:

The analyzed dosing formulations contained 99.3% to 103% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

One female in the 600 mg/kg/day group (No. 4508) was euthanized in extremis on Lactation Day 5 after findings of prolonged labor and clinical observations of pallor skin, red fur staining around the urogenital area or nose, pale skin on numerous body surfaces, slight salivation, increased respiration rate, a thin body, and/or cold to the touch during Lactation Days 0–5. Necropsy observations for this female included an enlarged and swollen liver, a pale white focus on the liver, pale discoloration of the pancreas, an enlarged spleen, and a small thymus gland. Microscopic findings of inflammation, necrosis, thrombosis, and/or bacteria in multiple organs including the heart, lung, liver, kidney, brain, and uterus were consistent with bacterial sepsis (possibly secondary to endometritis) which was deemed the cause of debility in this female and was not directly related to rhubafuran administration.

Test substance-related increases were noted for mean kidney weight in the 600 mg/kg/day group males and females, in mean liver weights in the 200 mg/kg/day group males and 600 mg/kg/day group males and females. The higher liver weights correlated to minimal hypertrophy microscopically which was noted in the 200 and 600 mg/kg/day groups while the increased kidney weights had no microscopic correlate. All organ weight and microscopic findings were considered adaptive and non-adverse.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic and reproductive toxicity

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Key result

Critical effects observed:

no

Conclusions:

Under the conditions of this screening study, based on a lack of adverse findings noted during the premating period or on neurobehavior, motor activity, and pathology parameters, a dose level
of 600 mg/kg/day (the highest dose level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female systemic toxicity when rhubafuran when administered orally by gavage to Crl:CD(SD) rats.

Executive summary:

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD TG 422 and in compliance with GLP to evaluate the potential toxic effects of the test substance when administered to rats for at least 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance. 

Animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days (Study Days 0–27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 (minimum of Study Days 0–38 or maximum of Study Days 0–60 based on breeding success).

 

The experimental study design was as follows:

Group	Test Substance	Dosage Level (mg/kg/day)^a	Dose Concentration (mg/mL)	Dose Volume (mL/kg)	Number of animals
					Males	Females
1	Vehicle control	0	0	5	10	10
2	Test item	100	20	5	10	10
3	Test item	200	40	5	10	10
4	Test item	600	120	5	10	10

^aNot corrected for salt, purity, and water content.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, thyroid hormones, clinical pathology, macroscopic findings, organ weights, and microscopic examinations.

 

Results

The analyzed dosing formulations were within the protocol-specified range of target concentrations for suspensions and were homogeneous.

One female in the 600 mg/kg/day group was euthanized in extremis on Lactation Day 5 following clinical observations of red fur staining around the urogenital area or nose, pale skin on numerous body surfaces, skin pallor, slight salivation, increased respiratory rate, a thin body, and/or body cold to the touch during Lactation Days 0–5. This female was also noted with difficulties during parturition, initially delivering 3 live and 3 dead pups followed by an additional 6 dead pups 2 days later. At necropsy, this female was noted with an enlarged and swollen liver, a pale white focus on the liver, pale discoloration of the pancreas, an enlarged spleen, and a small thymus gland. Microscopic findings of inflammation, necrosis, thrombosis, and/or bacteria in multiple organs including the heart, lung, liver, kidney, brain, and uterus were consistent with bacterial sepsis (possibly secondary to endometritis), which was deemed the cause of debility in this female, and was likely associated with prolonged labor (dystocia). This death was not considered to be directly related to rhubafuran administration.

All other F0 males and females survived to the scheduled necropsy. Clinical observations of salivation or evidence thereof (wet fur around the mouth and/or muzzle) was noted approximately 1 hour following dose administration for 1 and 4 F0 males in the 200 and 600 mg/kg/day groups, respectively, during Study Days 13–24 and for 2 and 7 surviving F0 females at 200 and 600 mg/kg/day, respectively, during Study Days 10–15, Gestation Days 0–9, and/or Lactation Days 1–13 compared to 0 instances in the control group. These salivation related clinical observations were considered test substance-related but not adverse because they did not persist to the daily examinations. Additionally, these observations may have been associated with unpleasant smell and/or taste of the test substance due to its olfactory potency. No other test substance-related clinical observations were noted.

There were no test substance-related effects on mean body weight, body weight gains, or food consumption for F0 males and females in the 100, 200, and 600 mg/kg/day throughout the treatment period, including during gestation and lactation for females. No test substance-related effects were noted for estrous cyclicity, reproductive performance (mating, fertility, and pregnancy indices and precoital intervals) at any dose level.

Prolonged labor was noted for the female that was euthanized in extremis at 600 mg/kg/day, however, this death was not considered directly related to rhubafuran administration due to the underlying evidence of bacterial sepsis in this female. There were no signs of dystocia in the 100 and 200 mg/kg/day groups or the remaining females in the 600 mg/kg/day group, and mean postimplantation loss, mean gestation length and gestation index in the 100, 200, and 600 mg/kg/day groups were unaffected by test substance administration.

Neurobehavior and motor activity for F0 males and females in the 100, 200, and 600 mg/kg/day groups were unaffected by test substance administration.

Mean hematology, coagulation, and serum chemistry parameters in the 100, 200, and 600 mg/kg/day group F0 males and females were unaffected by test substance administration. Differences from control were slight, not statistically significant, not observed in a dose-related manner, and/or had no clinical pathology or microscopic correlates and were considered a result of biological variation. Mean thyroid hormone concentrations (T3, T4, and TSH) for F0 males
was unaffected by test substance administration at all dose levels.

Administration of the test substance resulted in minimal hepatocellular hypertrophy in the 200 and 600 mg/kg/day groups that correlated to increased mean liver weights in the 200 mg/kg/day males and 600 mg/kg/day group males and females. In addition, there were increased mean kidney weights in the 600 mg/kg/day group males and females that did not have a microscopic correlate. All findings were considered adaptive to test substance administration and non-adverse.

A lower mean viability index was noted for F1 pups in the 600 mg/kg/day group (80.31% per litter) compared to the control group (95.82% per litter) and was below the minimum mean value for the equivalent parameter in the Charles River Ashland historical control data (84.5% per litter; version 2019.05). Although this difference was partially attributed to the euthanized in extremis female that had 9 dead pups prior to euthanasia, 3 other litters in this group had low individual viability indices between 60.0% and 84.6%. Therefore, a relationship between lower pup survival and rhubafuran administration could not be ruled out. No test substance-related abnormalities were noted for these unscheduled pups at necropsy. Mean viability index in the 100 and 200 mg/kg/day groups, and mean numbers of newborn pups, live newborn pups, percentage of males at birth, live birth index, and survival index at all dose levels were unaffected by test substance administration.

There were no test substance-related clinical observations or necropsy findings, and no test substance-related effects on mean absolute body weights, body weight gains, developmental parameters (absolute and normalized anogenital distance and nipple retention), thyroid hormone concentrations (T3, T4, and TSH), and absolute thyroid/parathyroid gland weights for F1 male and female pups at any dose level.

 

Conclusion

In conclusion, under the conditions of this screening study, based on a lack of adverse findings noted during the premating period or on neurobehavior, motor activity, and pathology parameters, a dose level of 600 mg/kg/day (the highest dose level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female systemic toxicity when rhubafuran when administered orally by gavage to Crl:CD(SD) rats. There were no effects on male or female reproductive indices at any dose level; therefore, 600 mg/kg/day was considered to be the NOAEL for F0 male and female reproductive toxicity.

Based on the lower mean viability index and F1 pup mortality noted in the 600 mg/kg/day group, a dose level of 200 mg/kg/day was considered to be the NOAEL for F1 neonatal toxicity.

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the rat is acceptable and satisfies the guideline requirement for an OECD 422 study in rats. 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Recent GLP study conducted according to OECD Guideline No 422 without any deviation (Klimisch score = 1).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A key study was available (CRL, 2021, rel.1, K).

In this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test performed according to OECD TG 422 and in compliance with GLP, animals were dosed via oral gavage once daily. Males were dosed for 14 days prior to mating and continuing throughout mating for a minimum of 28 days (Study Days 0–27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 (minimum of Study Days 0–38 or maximum of Study Days 0–60 based on breeding success).

 

The experimental study design was as follows:

Group	Test Substance	Dosage Level (mg/kg/day)^a	Dose Concentration (mg/mL)	Dose Volume (mL/kg)	Number of animals
					Males	Females
1	Vehicle control	0	0	5	10	10
2	Test item	100	20	5	10	10
3	Test item	200	40	5	10	10
4	Test item	600	120	5	10	10

^aNot corrected for salt, purity, and water content.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, neurobehavior, thyroid hormones, clinical pathology, macroscopic findings, organ weights, and microscopic examinations.

 

Results

The analyzed dosing formulations were within the protocol-specified range of target concentrations for suspensions and were homogeneous.

One female in the 600 mg/kg/day group was euthanized in extremis on Lactation Day 5 following clinical observations of red fur staining around the urogenital area or nose, pale skin on numerous body surfaces, skin pallor, slight salivation, increased respiratory rate, a thin body, and/or body cold to the touch during Lactation Days 0–5. This female was also noted with difficulties during parturition, initially delivering 3 live and 3 dead pups followed by an additional 6 dead pups 2 days later. At necropsy, this female was noted with an enlarged and swollen liver, a pale white focus on the liver, pale discoloration of the pancreas, an enlarged spleen, and a small thymus gland. Microscopic findings of inflammation, necrosis, thrombosis, and/or bacteria in multiple organs including the heart, lung, liver, kidney, brain, and uterus were consistent with bacterial sepsis (possibly secondary to endometritis), which was deemed the cause of debility in this female, and was likely associated with prolonged labor (dystocia). This death was not considered to be directly related to rhubafuran administration.

All other F0 males and females survived to the scheduled necropsy. Clinical observations of salivation or evidence thereof (wet fur around the mouth and/or muzzle) was noted approximately 1 hour following dose administration for 1 and 4 F0 males in the 200 and 600 mg/kg/day groups, respectively, during Study Days 13–24 and for 2 and 7 surviving F0 females at 200 and 600 mg/kg/day, respectively, during Study Days 10–15, Gestation Days 0–9, and/or Lactation Days 1–13 compared to 0 instances in the control group. These salivation related clinical observations were considered test substance-related but not adverse because they did not persist to the daily examinations. Additionally, these observations may have been associated with unpleasant smell and/or taste of the test substance due to its olfactory potency. No other test substance-related clinical observations were noted.

There were no test substance-related effects on mean body weight, body weight gains, or food consumption for F0 males and females in the 100, 200, and 600 mg/kg/day throughout the treatment period, including during gestation and lactation for females. No test substance-related effects were noted for estrous cyclicity, reproductive performance (mating, fertility, and pregnancy indices and precoital intervals) at any dose level.

Prolonged labor was noted for the female that was euthanized in extremis at 600 mg/kg/day, however, this death was not considered directly related to rhubafuran administration due to the underlying evidence of bacterial sepsis in this female. There were no signs of dystocia in the 100 and 200 mg/kg/day groups or the remaining females in the 600 mg/kg/day group, and mean postimplantation loss, mean gestation length and gestation index in the 100, 200, and 600 mg/kg/day groups were unaffected by test substance administration.

Neurobehavior and motor activity for F0 males and females in the 100, 200, and 600 mg/kg/day groups were unaffected by test substance administration.

Mean hematology, coagulation, and serum chemistry parameters in the 100, 200, and 600 mg/kg/day group F0 males and females were unaffected by test substance administration. Differences from control were slight, not statistically significant, not observed in a dose-related manner, and/or had no clinical pathology or microscopic correlates and were considered a result of biological variation. Mean thyroid hormone concentrations (T3, T4, and TSH) for F0 males
was unaffected by test substance administration at all dose levels.

Administration of the test substance resulted in minimal hepatocellular hypertrophy in the 200 and 600 mg/kg/day groups that correlated to increased mean liver weights in the 200 mg/kg/day males and 600 mg/kg/day group males and females. In addition, there were increased mean kidney weights in the 600 mg/kg/day group males and females that did not have a microscopic correlate. All findings were considered adaptive to test substance administration and non-adverse.

A lower mean viability index was noted for F1 pups in the 600 mg/kg/day group (80.31% per litter) compared to the control group (95.82% per litter) and was below the minimum mean value for the equivalent parameter in the Charles River Ashland historical control data (84.5% per litter; version 2019.05). Although this difference was partially attributed to the euthanized in extremis female that had 9 dead pups prior to euthanasia, 3 other litters in this group had low individual viability indices between 60.0% and 84.6%. Therefore, a relationship between lower pup survival and rhubafuran administration could not be ruled out. No test substance-related abnormalities were noted for these unscheduled pups at necropsy. Mean viability index in the 100 and 200 mg/kg/day groups, and mean numbers of newborn pups, live newborn pups, percentage of males at birth, live birth index, and survival index at all dose levels were unaffected by test substance administration.

There were no test substance-related clinical observations or necropsy findings, and no test substance-related effects on mean absolute body weights, body weight gains, developmental parameters (absolute and normalized anogenital distance and nipple retention), thyroid hormone concentrations (T3, T4, and TSH), and absolute thyroid/parathyroid gland weights for F1 male and female pups at any dose level.

 

Conclusion

In conclusion, under the conditions of this screening study, based on a lack of adverse findings noted during the premating period or on neurobehavior, motor activity, and pathology parameters, a dose level of 600 mg/kg/day (the highest dose level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female systemic toxicity when rhubafuran when administered orally by gavage to Crl:CD(SD) rats. There were no effects on male or female reproductive indices at any dose level; therefore, 600 mg/kg/day was considered to be the NOAEL for F0 male and female reproductive toxicity.

Based on the lower mean viability index and F1 pup mortality noted in the 600 mg/kg/day group, a dose level of 200 mg/kg/day was considered to be the NOAEL for F1 neonatal toxicity.

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the rat is acceptable and satisfies the guideline requirement for an OECD 422 study in rats. 

 

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

 

Self-classification:

In a recent GLP combined repeated dose toxicity study with the reproduction / developmental screening test (OECD guideline 422), based on a lack of adverse findings noted during the premating period or on neurobehavior, motor activity, and pathology parameters and based on no effects on male or female reproductive indices at any dose level , a dosage level of 600 mg/kg bw/day, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive and systemic toxicity of Rhubafuran when administered orally by gavage to Crl:CD(SD) rats.

Therefore the registered substance is not classified for repeated dose toxicity according to CLP Regulation (EC) No 1272 /2008 and UN GHS criteria.