4-Decenal, 9-hydroxy-5,9-dimethyl-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 September 2020 to 25 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 421 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

9-hydroxy-5,9-dimethyldec-4-enal

EC Number:

940-437-2

Cas Number:

926-50-1

Molecular formula:

C12H22O2

IUPAC Name:

9-hydroxy-5,9-dimethyldec-4-enal

Test material form:

liquid

Details on test material:

Apearance: Colorless to ale yellow liquid
Odour: Floral, Green, Lily-of-the-valley, fatty odour
Condition of storage: 15-25°C, protected from light
Expiry date: 09 April 2021

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Crl:WI(Han) 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC on 11 Aug and 20 Aug 2020
- Age at study initiation: 11-12 weeks old
- Weight at study initiation: 163 to 301 g
- Housing: On arrival, animals were group housed (up to 3 animals of the same sex) until cohabitation. During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study.
- Diet : PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum throughout the study.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system.
Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Animal enrichment: Animals were socially housed for psychological/environmental enrichment. During periods of individual hosing, animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
- Acclimation period: After receipt at the Testing Facility, the Crl:WI(Han)rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC
- Humidity (%): 30-70%.
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark.

IN-LIFE PERIOD: 11 August 2020 to 02 November 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

The vehicle, corn oil, was dispensed daily for administration to Group 1 control animals and preparation of the test substance formulations

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance dosing formulations were prepared based on Sponsor instructions at appropriate
concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (2°C to 8°C), protected from light, under nitrogen, until use. The dosing formulations were stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 0, 20, 80 and 160 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

DOSE VOLUME: 5 mL/kg bw

Details on mating procedure:

After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group.
Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples of all groups were collected for concentration analysis and Group 2 and 4 dose formulation samples were collected for analysis of homogeneity to the Charles River Ashland Analytical Chemistry Department. Analyses were performed by a gas chromatography method with flame ionization detection, using a validated analytical procedure.
Test substance formulations have been previously shown to be stable and homogeneous over the
range of concentrations used on this study. Therefore, stability and resuspension homogeneity of test substance formulations will not be assessed on this study.

Duration of treatment / exposure:

Males : 14 days prior to mating and continuing throughout mating for a minimum of 28 days
Females: 14 days prior to mating and continuing throughout mating, gestation and lactation until 1 day prior to scheduled euthanasia. Females with no evidence of mating were dosed through the day prior to euthanasia.
The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Frequency of treatment:

The test substance and vehicle were administered as a single daily oral gavage dose at approximately the same time each day.

Details on study schedule:

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. Females not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (vehicle control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 (test item)

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Group 3 (test item)

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Group 4 (test item)

No. of animals per sex per dose:

10 animals/sex/dose

The number of animals was based on the OECD Guideline for the Testing of Chemicals: Guideline 421, Reproduction/Development Toxicity Screening Test, 29 July 2016, 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 fail to exhibit normal 4-5 day estrous cycles (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 test substance-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 route of administration was oral (gavage) because this is a potential route of exposure to humans. Historically, this route has been used extensively for studies of this nature.

The dose levels were determined from results of previous studies and will be provided by the Sponsor Representative. Dose selection for this study was based on a previous 28-day repeat dose study in Wistar Han rats (Study Number D77438) in which dose levels up to 800 mg/kg/day were generally well tolerated. At 800 mg/kg/day, males and females were noted with clinical observations of salivation postdosing and test substance related increased liver weights (males and females) and kidney weights (females). Microscopic observations of minimal centrilobular hepatocellular hypertrophy was noted in males and females at 800 mg/kg/day and increased hyaline droplets were noted in males at 800 mg/kg/day. The liver centrilobular hepatocellular hypertrophy was noted as fully reversable after the recovery period, and the findings of increased hyaline droplets were not considered adverse. There was no test substance related mortality or effects on body weights or food consumption.
Based on these results 0, 100, 400 and 800 mg/kg/day dose levels were selected. The high-dose level was expected to produce some toxicity, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal toxic effects. The low-dose level was expected to produce no observable indications of toxicity.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily throughout the study, prior to dosing. On dosing days, clinical observations were also recorded approximately 2 hours postdose.
The animals were removed from the cage, and a detailed clinical observation was performed.
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: Animals were weighed individually 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, 3, 7, 10, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 10, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): YES
Food consumption was quantitatively measured weekly until cohabitation. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 3, 7, 10, 14, 17, and 21 and Lactation Days 1, 4, 7, 10, and 13. Food consumption was not recorded for females with no evidence of mating following the end of the breeding period.

THYROID HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: At study day 28 for males and at lactation day 13 for females. Blood samples for thyroid hormone analyses were collected from a jugular vein into tubes without anticoagulants.
- Processing: Blood samples were maintained at room temperature and allowed to clot for at least 30 minutes. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain -70°C. Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were conducted using validated ultra high-performance liquid chromatography with dual mass spectroscopy (UHPLCMS/MS) assays.
- Analysis: Thyroxine (Total T4).

OTHER:
- Parturition Observations and Gestation Length: The day parturition was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). During the period of expected parturition, females were observed 3 times daily for initiation and completion of parturition and for dystocia or other difficulties. All females were allowed to deliver naturally. Beginning on the day parturition was initiated, the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed.

Oestrous cyclicity (parental animals):

For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. The slides were microscopically examined to determine the stage of the estrous cycle.
The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of mating).
Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

Not examined

Litter observations:

STANDARDISATION OF LITTERS
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 and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Viability: Litters 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.
- Clinical observations: The animals were removed from the cage, and a detailed clinical observation was performed on PND 1, 4, 7, 10, and 13.
- Litter size: A daily record of litter size was maintained.
- Total litter loss: Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia.
- Sex ratio: Pups were individually sexed on PND 0 or 1, and on PND 4 and 13.
- Individual offspring bodyweights: Pups were weighed individually on PND 1, 4, 7, 10, and 13.
- Ano-genital distance: The anogenital distance of all pups was measured on PND 1 and 4. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.
- Nipple/ areolae count: On PND 13, all male pups were evaluated for the presence of nipples/areolae. The number of nipples was recorded.

THYROID HORMONE ANALYSIS: Yes
- Time schedule for collection of blood: At least 2/litter at PND 4 and 2/sex/litter at PND 13. Blood samples for thyroid hormone analyses were collected via cardiac puncture from animals anesthetized with isoflurane into tubes without anticoagulants.
- Processing: Blood samples were maintained at room temperature and allowed to clot for at least 30 minutes. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain -70°C. Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were conducted using validated ultra high-performance liquid chromatography with dual mass spectroscopy (UHPLCMS/MS) assays.
- Analysis: Thyroxine (Total T4).

Postmortem examinations (parental animals):

SACRIFICE
No animals died during the course of the study. All surviving animals, including females with total litter loss, were euthanized by carbon dioxide inhalation.
See terminal procedures table below (Table 7.8/01).

GROSS 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 former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. 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.

Adrenal glands
Brain
Epididymides (paired organs weighed separately)
Heart
Kidneys
Liver
Ovaries (with oviducts)
Pituitary gland
Prostate gland
Seminal vesicle (with coagulating gland and fluid)
Spleen
Testes (paired organs weighed separately)
Thymus gland
Thyroids (with parathyroids), after fixation

Representative samples of the tissues identified below were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.

Brain
Coagulating gland
Kidneys
Liver
Mammary glands
Ovaries and oviduct (2)
Pituitary gland
Prostate gland
Seminal vesicles (2)
Testes with epididymides (2), placed in modified Davidson’s solution.
and vas deferens
Uterus with cervix and vagina
Thyroid (with parathyroids)
All gross lesions (Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible)

Tissue trimming was performed at the Testing Facility. Tissues identified in the table from all animals in the control and high-dose groups, as well as gross lesions and kidneys (males only) from all groups, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin for examination. In addition, PAS staining was used for the testes and epididymides. Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified in the table for microscopic examination were evaluated from all animals in the control and high-dose groups.

Postmortem examinations (offspring):

SACRIFICE
Intact offspring that were found dead during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead after PND 4. On PND 4, surviving animals were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded.
On PND 13, surviving animals were euthanized via an intraperitoneal injection of sodium
pentobarbital.
See terminal procedures table below (Table 7.8/02).

GROSS NECROPSY
On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. All other animals were discarded without examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
Thyroid with parathyroids (if present) were weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia. Organ weights were not recorded for animals found dead.
Representative samples of the thyroid were collected from 1 pup/sex/litter at the scheduled euthanasia and preserved in 10% neutral buffered formalin.

Statistics:

Data collected during the pre-dose 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, 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 table 7.8/03, when possible, but will exclude any group with less than 3 observations.

Reproductive indices:

Means, standard deviations, ratio, percentages, numbers, and/or incidences were reported as
appropriate by dataset.
The following parental indices and natural delivery/reproductive parameters were reported, as
appropriate:
Gestation Length: The gestation length is calculated from GD 0 to the day the first pup is observed.
Female Mating Index = Number of Females with Evidence of Mating (or no confirmed mating date and pregnant) / Number of Females Paired
Female Fertility Index = Number of Pregnant Females / Number of Females with Evidence of Mating (or no confirmed mating date and pregnant)
Female Pregnancy Index = Number of Pregnant Females / Number of Females Paired
Male Mating Index = Number of Males with Evidence of Mating (or female partner confirmed pregnant) / Number of Males Paired
Male Fertility Index = Number of Males Impregnating a Female / Number of Males with Evidence of Mating (or female partner confirmed pregnant)
Male Pregnancy Index = Number of Males Impregnating a Female / Number of Males Paired
Gestation Index: Percentage of pregnancies that result in birth of live litters
Number of Animals with Live Offspring x 100 / Number of Animals Pregnant

Offspring viability indices:

Number of implantation sites;
Number of offspring per litter: Live and dead pups;
General condition of dam and litter during the postpartum period;
Live Birth Index: Percentage of pups born alive;
Number of Live Newborn Pups x 100 / Number of Newborn Pups;
Viability Index: Percentage of pups born that survive 4 days postpartum;
Number of Live Pups on Day 4 Postpartum x 100 / Number of Liveborn Pups;
Lactation Index: Percentage of pups that survive 13 days postpartum;
Number of Live Pups on Day 13 Postpartum x 100 / Number of Live Pups on Day 4 Postpartum.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Abnormal breathing sounds was noted in 2 females and wet fur around the mouth was noted for
4 females in the 800 mg/kg/day group at the 2-hour post-dosing examinations during Lactation
Days 2-10. These findings were considered test substance-related; however, these findings were
transient, did not persist the daily examinations, and with the exception of Female No. 4502,
were noted only once for each affected female and as such were not considered adverse. No
other test substance-related clinical observations were noted for males or females at any dosage levels. Noted clinical observations occurred in similar incidence in the control group and/or in a
manner that was not dose responsive.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Single females in each of the control (Female No. 1507) and 400 mg/kg/day (Female No. 3509) groups had total litter losses on Lactation Day 1 and were euthanized. All other males and females in the control, 100, 400, and 800 mg/kg/day groups survived to the scheduled euthanasia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALES
Mean body weight gains in the 800 mg/kg bw/day group males were generally slightly lower than the control group throughout the dosing period; differences were not statistically significant. As a result, mean absolute body weights in the 800 mg/kg/day group were 3.0% lower (not statistically significant) than the control group on Study Day 28. However, due to the small magnitude of difference from the control group, the difference was considered test substance-related but non-adverse. Mean body weights and body weight gains in the 100 and 400 mg/kg/day group males were unaffected by test substance administration throughout the study.

FEMALES
Mean body weights and body weight gains in the 100, 400, and 800 mg/kg/day group females were unaffected by test substance administration during the premating period, the gestation and the lactation period. None of the differences from the control group were statistically significant.
[TBC]

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALES
Mean food consumption, evaluated as g/animal/day, in the 100, 400, and 800 mg/kg/day group males was similar to that in the control group throughout the premating period. No statistically significant differences were observed.

FEMALES
Mean food consumption, evaluated as g/animal/day, in the 100, 400, and 800 mg/kg/day group females was unaffected by test substance administration during the premating, the gestation and the lactation period. None of the differences from the control group were statistically significant during premating, but statistically significantly higher mean food consumption was noted for females in the 400 mg/kg/day group during Gestation Days 3-7; however, this did not occur in a dose-related manner and was transient. No other 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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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):

Test substance-related accumulation of hyaline droplets in the kidney was characterized by large spherical to amorphous lightly eosinophilic intracellular globules, primarily in the proximal convoluted tubules. This finding was considered non-adverse given the limited severity and lack
of other changes in the kidney.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and
treated animals and, therefore, were considered unrelated to administration of Mahonial.
See text table 19 in the full study report attached.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related lower T4 hormone levels were noted for males in the 800 mg/kg/day group. T4 levels were 28.7% lower than the concurrent control group. This was considered non-adverse due to a lack of corresponding macroscopic or microscopic findings or effects on thyroid gland weights. T4 levels in the 100 and 400 mg/kg/day group males were unaffected by test substance administration.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also
similar to the control group value. None of these differences were statistically significant.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. All males sired a litter and all females were gravid.
Mean gestation lengths in the 100, 400, and 800 mg/kg/day groups were similar to those in the
control group. No statistically significant differences were noted. No signs of dystocia were
noted in these groups. See text table 17 in the full study report attached.

Details on results (P0)

Test substance-related observations of abnormal breathing sounds and wet fur around the mouth
were noted for 2 or 4 (of 10) F0 females in the 800 mg/kg/day group at the 2-hour postdosing
examinations during Lactation Days 2-10. These findings were considered test substance-related;
however, these findings were transient, did not persist the daily examinations, and/or were noted
only once for each affected female, and as such were not considered adverse. No other test
substance-related clinical observations were noted for males or females at any dosage levels.
Mean body weight gains in the 800 mg/kg/day group F0 males were generally slightly lower than
the control group throughout the dosing period, which resulted in mean absolute body weights
that were 3.0% lower than the control group on Study Day 28. However, due to the small magnitude of difference from the control group, the differences were considered test substance-related but non-adverse. No other effects on body weights or body weight gains were noted for F0
males or females at any dosage level.
Test substance-related lower T4 levels were noted for F0 males in the 800 mg/kg/day group.
T4 levels were 28.7% lower than the concurrent control group. This was considered non-adverse
due to a lack of corresponding macroscopic or microscopic findings or effects on thyroid gland
weights. T4 levels in the 100 and 400 mg/kg/day group males were unaffected by test substance
administration.
Test substance-related higher mean liver weights were noted in the 400 mg/kg/day group
F0 males and in the 800 mg/kg/day group, in both sexes, but did not have any microscopic
correlates. Test substance-related higher mean kidney weights were noted in the 100 and
400 mg/kg/day group males and in the 800 mg/kg/day group in both sexes and correlated with
accumulation of hyaline droplets in males at all dosage levels. This finding was minimal to mild
in severity and considered non-adverse.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations)
of all F1 pups in this study was unaffected by test substance administration.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Six (1), one (1), two (2), and zero (0) pups (litters) in the control, 100, 400, and 800 mg/kg/day groups, respectively, were found dead or euthanized in extremis.
Zero (0), two (1), one (1), and four (3) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
No internal findings were noted at the necropsies of pups that were found dead or euthanized in extremis in the test substance-treated groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male and female pup body weights in all test substance treated groups were generally similar to the control group on PND 1. Female pup body weight gains in the 800 mg/kg/day
group were generally slightly lower than the control group during PND 1-13, resulting in mean
body weights that were 5.19% lower than the control group on PND 13; differences were not
statistically significant. This was considered test substance-related but non-adverse due to the
small magnitude of difference from the control group.
Mean male and female pup body weights and body weight changes in the 100 and
400 mg/kg/day groups and male pup body weights and body weight changes at 800 mg/kg/day
were unaffected by test substance administration.

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:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on male or female anogenital distances when measured on PND 1 and 4. Male pup anogenital distance normalized to cubed root of body
weight in the 100, 400, and 800 mg/kg/day groups was statistically significantly higher than the
control group on PND 1 and similar to the control group on PND 4. Male and female pup
absolute anogenital distance and female normalized anogenital distance were similar to the control group. As such, differences in normalized anogenital distances on PND 1 in the 100, 400,
and 800 mg/kg/day males, in the absence of statistically significant effects on absolute anogenital distance or effects on absolute or normalized anogenital distance on PND 4, were considered natural variation, and unrelated to test substance administration.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test
substance when evaluated on PND 13. The test substance-treated group values were not
statistically different from the control group values.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on thyroid/parathyroid weights in the F1 males and
females at any dosage level on PND 13. Differences from the control group were considered to
be the result of normal biological variation and were not considered to be of toxicological
significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No internal findings were noted at the necropsies of pups that were found dead or euthanized in extremis in the test substance-treated groups and no internal findings were noted at the necropsy of pups euthanized on PND 13.

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on T4 values in the F1 males and females at any
dosage level on PND 13. Differences from the control group were considered to be the result of
normal biological variation and were not considered to be of toxicological significance.
The mean number of pups born, live litter size and the percentage of males at birth in the 100,
400, and 800 mg/kg/day groups were similar to the control group values. Postnatal survival in
the 100, 400, and 800 mg/kg/day groups were unaffected by test substance administration.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

In the F1 generation, female pup body weight gains in the 800 mg/kg/day group were generally slightly lower than the control group during PND 1-13, resulting in mean body weights that were
5.19% lower than the control group on PND 13. This difference was considered test substancerelated but non-adverse due to the small magnitude of difference from the control group.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects oberved

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

The analyzed dosing formulations contained 104% to 112% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and was homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1). Result of the analyses of dosing formulations are summarized below:

 

Table 7.8/04: results of homogeneity analyses

	Group 2 (20 mg/mL)	Group 4 (160 mg/mL)
Homogeneity assessment of the 31 Aug 2020 formulations
Mean concentration (mg/mL)	21.2	171
RSD (%)	1.6	1.4
Mean % of target	106	107

 

 

Table 7.8/05: results of concentration analyses

Mean concentration, mg/mL (% of target)
Date of preparation	Group 2 (20 mg/mL)	Group 3 (80 mg/mL)	Group 4 (160 mg/mL)
31 Au 2020	21.4 (107)	84.2 (105)	170 (106)
14 Sep 2020	20.8 (104)	85.7 (107)	168 (105)
20 Oct 2020	21.2 (106)	88.6 (111)	179 (112)

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, based on the lack of any adverse effects at any
dosage level, a dosage level of 800 mg/kg/day, the highest dosage level evaluated, was
considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive and
systemic toxicity and F1 neonatal toxicity of Mahonial when administered orally by gavage to
Crl:WI(Han) rats.

Executive summary:

This study is a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, via daily oral gavage of the test item from conception through day 13 of postnatal rats life.

Three groups of ten male and ten female rats received the test item at dose level of 100, 400 and 800 mg/kg bw/day. Males were dosed for 14 days prior to mating and continuing through 1 day prior to euthanasia (Study Days 0–27). Females were dosed for 14 days prior to mating and continuing through Lactation Day 12.. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle control, corn oil, throughout the same period. 

An additional ten males were assigned to each of the Control and high dose groups and received either the control or treated diet for 13 consecutive weeks. These animals constituted the Toxicity phase of the study and were used for sperm analysis and histopathology of testes and epididymides at the end of the treatment period.

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

 

No test substance-related effects were noted on F0 survival, food consumption, reproductive performance (mating, fertility, and pregnancy), estrous cyclicity, or parturition, and no test substance-related gross findings were noted for F0 animals at any dosage level. No test substance-related effects were noted on F1 litter viability and survival, anogenital distance, areolae/nipple anlagen retention, thyroid hormones and organ weights, and no test substance-related clinical or macroscopic findings were noted at any dosage level.

Test substance-related observations of abnormal breathing sounds and wet fur around the mouth were noted for 2 or 4 (of 10) F0 females in the 800 mg/kg/day group at the 2-hour postdosing examinations during Lactation Days 2-10. These findings were considered test substance-related; however, these findings were transient, did not persist the daily examinations, and/or were noted only once for each affected female, and as such were not considered adverse. No other test
substance-related clinical observations were noted for males or females at any dosage levels.

Mean body weight gains in the 800 mg/kg/day group F0 males were generally slightly lower than the control group throughout the dosing period which resulted in mean absolute body weights that were 3.0% lower than the control group on Study Day 28. However, due to the small magnitude of difference from the control group, the differences were not considered adverse. No other effects on body weights or body weight gains were noted for F0 males or females at any dosage level.

Test substance-related lower T4 levels were noted for F0 males in the 800 mg/kg/day group. T4 levels were 28.7% lower than the concurrent control group. This difference was considered non-adverse due to a lack of corresponding macroscopic or microscopic findings or any effects on thyroid gland weights. T4 levels in the 100 and 400 mg/kg/day group males were unaffected by test substance administration.

Test substance-related higher mean liver weights were noted in the 400 mg/kg/day group F0 males and in the 800 mg/kg/day group, in both sexes, but did not have any microscopic correlates. Test substance-related higher mean kidney weights were noted in the 100 and 400 mg/kg/day group males and in the 800 mg/kg/day group in both sexes and correlated with accumulation of hyaline droplets in males at all dosage levels. This finding was minimal to mild in severity and considered non-adverse.

In the F1 generation, female pup body weight gains in the 800 mg/kg/day group were generally slightly lower than the control group during PND 1-13, resulting in mean body weights that were 5.19% lower than the control group on PND 13. This difference was considered test substance-related but non-adverse due to the small magnitude of difference from the control group.

 

Under the conditions of this screening study, based on the lack of any adverse effects at any dosage level, a dosage level of 800 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive and systemic toxicity and F1 neonatal toxicity of Mahonial when administered orally by gavage to Crl:WI(Han) rats.