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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

OECD 421, GLP Study (2021) :

NOAEL parental toxicity >= 250 mg/kg bw/d

NOAEL reproductive toxicity >= 250 mg/kg bw/d

NOAEL neonatal toxicity >= 250 mg/kg bw/d

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 June to 21 August 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
- Route of administration
The oral (gavage) route of exposure was chosen to comply with regulatory requirements and as historically, this route has been used extensively for studies of this nature, although the most likely route of human exposure during manufacture, handling or use of the test item is the dermal route.

- Basis for dose level selection:
Dose selection for this study was based on a previous rat 28-day repeat dose study (Braun et al., 2000) in which Javanol was administered at doses of 20, 100, and 500 mg/kg/day. Javanol induced transient reduction of food consumption, changes in clinical biochemistry parameters, increased weights in liver at 500 mg/kg/day for both sexes, increased kidney weights in males at 500 mg/kg/day, and decreased thymus weight in males at 100 and 500 mg/kg/day. Associated microscopic findings were noted in the kidneys of males at 500 mg/kg/day.
Based on these results, dose levels of 0, 50, 100, and 250 mg/kg/day were selected for the current study. 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.
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
On 09 Jun and 18 Jun 2020, Crl:WI(Han) female and male rats, respectively, were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 11–12 weeks old and weighed between 146 to 198 g for females and 295 and 362 g for males at the initiation of dosing.

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. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals is based on the OECD Guideline for the Testing of Chemicals: Guideline 421, Reproduction/Development Toxicity Screening Test, 29 Jul 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 failed 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 treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

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 at extremes of body weight range or females 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 and discarded.
Sex:
male/female
Details on test animals or test system and environmental conditions:
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.
Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dose level, and sex. Cages were arranged on the racks in group order. Where possible, control group animals were housed on a separate rack from the test substance-treated animals.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

Environmental conditions:
Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained.

Food:
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:
For enrichment, animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.

Veterinary care:
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.
Route of administration:
oral: gavage
Vehicle:
other: 10% dimethyl sulfoxide (DMSO) and 70% polyethylene glycol (PEG) 300 in deionized water
Details on exposure:
Preparation of Vehicle:
The vehicle, 10% dimethyl sulfoxide (DMSO) and 70% polyethylene glycol (PEG) 300 in deionized water, was prepared weekly and stored refrigerated (target of 5°C), protected from light. For administration to Group 1 control animals, an adequate amount of the vehicle was dispensed into daily aliquots, which were stored refrigerated (target of 5°C), protected from light, until use. The vehicle was stirred continuously during dosing.

Preparation of Test Substance:
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, until use. The dosing formulations were stirred continuously during dosing.

Administration of Test Materials
The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia (for a minimum of 28 days). Females were dosed for 14 days prior to mating and continuing through Lactation Day 12. Females with no evidence of mating were dosed through the day prior to euthanasia. All animals were dosed at approximately the same time each day.
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.
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 were collected for analysis as follows:
- Concentration => All groups
- Homogeneity => Groups 2 and 3

Analyses described below were performed by a gas chromatography method with flame ionization detection using a validated analytical procedure.

- Concentration analysis:
Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration, with individual sample concentrations within ± 20% of the target concentration. After acceptance of the analytical results, backup samples were discarded.

- Homogeneity and Stability Analysis:
Test substance formulations have been previously shown to be stable and homogeneous over the range of concentrations used on this study (Akalkotkar, 2020, 00810014). Therefore, stability and resuspension homogeneity of test substance formulations was not assessed on this study.
Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each concentration was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
Duration of treatment / exposure:
Males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia (for a minimum of 28 days). Females were dosed for 14 days prior to mating and continuing through Lactation Day 12. Females with no evidence of mating were dosed through the day prior to euthanasia. All animals were dosed at approximately the same time each day.
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.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Vehicle Control)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 males and 10 females per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- 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 at extremes of body weight range or females 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 and discarded.
Positive control:
None
Parental animals: Observations and examinations:
- Viability:
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.

- Observations:
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 2 hours postdose.
During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

- Body Weights:
Animals were weighed individually 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, and 13.

- 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 on Lactation Days 1, 4, 7, 10, and 13.

- Breeding Procedures:
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.

- Parturition
The day parturition was completed 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 live pups were recorded. Individual gestation length was calculated using the date delivery was completed.
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 determined
Litter observations:
- Viability:
Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

- Observations:
A detailed clinical observation was performed on PND 1, 4, 7, 10, and 13.

- Sex Determination:
Pups were individually sexed on PND 0 or 1, and on 4 and 13.

- Body Weights:
Pups were weighed individually on PND 1, 4, 7, 10, and 13.

- Anogenital 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 (Gallavan et al., 1999).

- Assessment of Areolas/Nipple Anlagen Retention:
On PND 13, all male pups were evaluated for the presence of nipples/areolae (Gray et al., 1999). The number of nipples was recorded.
Postmortem examinations (parental animals):
- Unscheduled Deaths:
A necropsy was conducted for animals that died on study, and specified tissues were saved.
- Scheduled Euthanasia:
All surviving animals, including females that failed to deliver or with total litter loss, 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 the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).

- Organ Weights:
The organs identified in the list below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or 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: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Pituitary gland, Prostate gland, Seminal vesicles (with coagulating gland and fluid), Spleen, Testes, Thymus gland, Thyroids with parathyroids.

- Tissue Collection and Preservation:
Representative samples of the tissues identified below were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.
List of tissues: Brain, Coagulating glands, Kidneys, Liver, Mammary glands, Ovaries and oviducts, Pituitary gland, Prostate gland, Seminal vesicles, Testes with epididymides and vas deferens, Thyroids (with parathyroids, if present), Uterus with cervix and vagina, All gross lesions.

- Histology:
Tissues from all animals 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.

- Histopathology:
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues for microscopic examination were evaluated from all animals in the control and high-dose groups and from all animals found dead. Gross lesions were examined from all groups.

- Thyroid Hormone Analysis:
Blood samples were collectedfor thyroid hormone analyses (T4) from the jugular vein of males (group 1-4) into tubes without anticoagulants. Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay (Lucarell, 99764, 2017).
Postmortem examinations (offspring):
- Unscheduled Deaths:
A necropsy was conducted for animals that died on study, and specified tissues were retained.
If necessary, for humane reasons, animals were euthanized as per Testing Facility SOPs). These animals underwent necropsy, and specified tissues were retained.
Intact offspring that were found dead or euthanized for humane reasons during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Findings were recorded as developmental variations or malformations, as appropriate. Representative specimens with malformations were preserved in the 10% neutral buffered formalin.

- Scheduled Euthanasia
On PND 13, surviving animals were euthanized via an intraperitoneal injection of sodium pentobarbital.

- 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.

- Organ Weights:
Thyroid (with parathyroids, if present) was weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis.

- Tissue Collection and Preservation:
Representative samples of the Thyroid (with parathyroids, if present) were collected from 1 pup/sex/litter at the scheduled euthanasia and preserved in 10% neutral buffered formalin.

- Thyroid Hormone Analysis:
Blood samples for thyroid hormone analyses (T4) were collected via cardiac puncture from animals anesthetized with isoflurane into tubes without anticoagulants.
Samples were collected from 2 pups/sex/litter of groups 1-4 at PND 13. Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were conducted using a validated UHPLCMS/MS assay (Lucarell, 99764, 2017).
Statistics:
See in other information section
Reproductive indices:
Gestation Length, Female Mating Index, Female Fertility Index, Female Pregnancy Index, Male Mating Index, Male Fertility Index, Male Pregnancy Index, Gestation Index, Number of implantation sites.
Offspring viability indices:
Number of offspring per litter, Live Birth Index, Viability Index, Survival Index.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related clinical observations were noted during the generation at the daily examinations. Wet fur around the mouth was noted for 7 females in the 250 mg/kg/day group at 2 hours postdosing during Lactation Days 2–12; however, this finding was transient, did not persist the daily examinations, and was only noted on 1–2 days for each affected female.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test substance-related effects on survival at any dose level.
One male (No. 1009) in the control group and 1 female (No. 2507) in the 50 mg/kg/day group were found dead on Study Day 10. Male No. 1009 was noted with abnormal breathing sounds during Study Days 7–9. Macroscopic findings of an esophageal perforation and/or clear fluid accumulation in the thoracic cavity were noted for both animals that were found dead, indicating that these deaths were the result of dosing errors and were not test substance-related.
Two females (Nos. 1501 and 4505) in the control and 250 mg/kg/day groups, respectively, had total litter losses and were subsequently euthanized on Lactation Days 1 and 3, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES:
Mean body weight gains in the 100 and 250 mg/kg/day group F0 males were generally lower than the control group throughout the dosing period; differences were statistically significant during Study Days 3–7 at 250 mg/kg/day and during Study Days 10–13 at both dose levels. As a result, statistically significantly lower mean body weight gains were noted in these groups when the overall premating period (Study Days 0–13) and entire dosing period (Study Days 0–28) were evaluated. Despite the decrements in body weight gain, mean absolute body weights in the 100 mg/kg/day group were generally comparable to the control group (within 5%) throughout the study and lower mean absolute body weights in the 250 mg/kg/day group (5.0% to 6.9% lower vs. controls) during Study Days 13-28 lacked statistical significance. Therefore, the test substance-related decrements in body weight gain at 100 and 250 mg/kg/day were not considered adverse.
Mean body weights and body weight gains in the 50 mg/kg/day group males were unaffected by test substance administration throughout the study. Mean body weight gain in this group was statistically significantly lower than the control group for the overall premating period (Study Days 0–13), but was comparable to the control group for the overall dosing period and mean absolute body weights were unaffected throughout the study. Therefore, the lower mean body weight changes at 50 mg/kg/day were considered transient and not test substance-related.

FEMALES:
- Twice Weekly:
Mean body weights and body weight gains in the 50, 100, and 250 mg/kg/day group females were unaffected by test substance administration during the premating period. None of the differences from the control group were statistically significant.
- Gestation:
Mean maternal body weight gains in the 250 mg/kg/day group were generally lower than the control group throughout gestation, including for the overall gestation dosing period (Gestation Days 0–20); the difference was statistically significant during Gestation Days 0–4. As a result, mean absolute body weight in this group was 4.7% lower (not statistically significant) than the control group on Gestation Day 20. These changes were considered test substance-related but nonadverse, due to the minimal impact on mean absolute body weight.
Mean body weights and body weight gains in the 50 and 100 mg/kg/day groups were unaffected by test substance administration during gestation.
- Lactation:
Mean body weights and body weight gains in the 50, 100, and 250 mg/kg/day groups were unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
MALES:
Mean food consumption in the 250 mg/kg/day group males was statistically significantly lower than the control group during Study Days 0-3, and remained slightly lower than the control group throughout the remainder of the premating dosing period (Study Days 3–13). These changes corresponded to lower mean body weight gains noted in this group throughout the study and were considered test substance-related, but nonadverse due to the minimal impact on mean absolute body weight.
Mean food consumption in the 50 and 100 mg/kg/day group males was similar to that in the control group throughout the premating dosing period.

FEMALES:
- Twice Weekly:
Mean food consumption in the 50, 100, and 250 mg/kg/day group females was unaffected by test substance administration during the premating period. None of the differences from the control group were statistically significant.
- Gestation:
Mean maternal food consumption in the 250 mg/kg/day group was lower than the control group throughout gestation; differences were generally statistically significant, including for the overall gestation dosing period (Gestation Days 0–20). These changes corresponded to the test substance-related lower mean body weight gains noted in this group throughout gestation, but were considered nonadverse due to the minimal impact on mean absolute body weight.
Mean food consumption in the 50 and 100 mg/kg/day groups was unaffected by test substance administration during gestation.
- Lactation:
Mean maternal food consumption in the 50, 100, and 250 mg/kg/day groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
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:
no effects observed
Description (incidence and severity):
There were no test substance-related microscopic findings for F0 males or females.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related microscopic findings for F0 males or females.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis:
Mean T4 concentrations in the 100 and 250 mg/kg/day group F0 males were statistically significantly lower (18.4% and 25.6%, respectively) than the control group. The change at 250 mg/kg/day was accompanied by higher liver and thyroid weights; these organ weight changes lacked microscopic correlates. Disruption of thyroid function has been shown to be the result of hepatic disposition of thyroid hormone (McClain et al., 1989), and the higher liver weights at 250 mg/kg/day may be indicative of increased metabolism and excretion of T4. Given this potential mode of action and the lack of histopathological findings in the thyroid at 250 mg/kg/day or other evidence of endocrine-disruption, the lower mean T4 concentrations at 100 and 250 mg/kg/day were not considered adverse.
There were no test substance-related effects on thyroid hormone values in the F0 males in the 50 mg/kg/day group. Differences from the control group were slight and not statistically significant.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean number and length of estrous cycles in the test substance-treated groups were also similar to the control group values. None of these differences were statistically significant.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on reproductive performance were observed at any dose level. No statistically significant differences were noted between the control and test substance-treated groups. One1, 1, 0, and 1 mating pairs in the control, 50, 100, and 250 mg/kg/day groups, respectively, did not produce a litter.
The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value.

Female Nos. 1501 and 4505 in the control and 250 mg/kg/day groups, respectively, had a total litter loss and were subsequently euthanized on Lactation Days 1 and 3, respectively. As a result, low viability indices (survival from PND 0–4) were noted in the control and 250 mg/kg/day groups (86.11% and 87.78%, respectively). However, there were no test substance-related effects on postnatal survival at any dosage level (see Section 8.3.1.).
Mean gestation lengths and gestation index in the 50, 100, and 250 mg/kg/day groups were similar to the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
The mean postimplantation loss (unaccounted-for sites) and implantation sites in the 50, 100, and 250 mg/kg/day groups were similar to the control group values.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test-item related adverse effects observed up to the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
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.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Three (3), 3(3), 1(1), and 1(1) pups (litters) in the control, 50, 100, and 250 mg/kg/day groups, respectively, were found dead or euthanized in extremis. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead or euthanized in extremis.
The total number of newborn pups, number of live newborn pups, and the percentage of males at birth in the 50, 100, and 250 mg/kg/day groups were similar to the control group values. Postnatal survival, including live birth index (survival from PND 0), viability index (survival from PND 0–4), and survival index (survival from PND 4–13), in the 50, 100, and 250 mg/kg/day groups were unaffected by test substance administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male and female birth weights (PND 1) in the 250 mg/kg/day group were slightly lower (3.19% and 4.66%, respectively; not statistically significant) compared to the control group. These pup weights were comparable to the slightly lower mean maternal body weights for females in the 250 mg/kg/day group on Gestation Day 20, which were attributable to the lower mean maternal body weight gains and maternal food consumption during gestation. Thereafter, during PND 1–10, lower mean male and female pup body weight gains were observed for the 250 mg/kg/day group; differences were occasionally statistically significant versus the control group. Correspondingly, increasingly lower (but not statistically significant) mean pup body weights were observed in this group from PND 4 through PND 10; male and female body weights were (12.34% and 11.96%, respectively) lower (not statistically significant) than the control group on PND 10. During PND 10–13, mean body weight gains for the 250 mg/kg/day group were comparable to the control group; however, due to the early deficits in body weight gain, absolute mean body weights at termination (PND 13) remained 10.65% and 10.60% lower (not statistically significant) than the controls for males and females, respectively, and mean body weights for males and females were outside the range of the Charles River Ashland historical control data on PND 10 and 13. While the early deficits in pup body weight gain in the 250 mg/kg/day group are likely test substance-related, there are no clear signs of adverse effects, given the evidence for recovery during the last interval prior to euthanasia (PND 10–13), and the absence of any other evidence of developmental toxicity (live birth index, pup birth weights, number of pups born, pup postnatal survival, etc.) at this dose level.

Mean male and female pup body weights and body weight changes in the 50 and 100 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period (PND 1–13).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
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):
no effects observed
Description (incidence and severity):
The anogenital distances (absolute and relative to the cube root of pup body weight) in the 50, 100, and 250 mg/kg/day groups on PND 1 and 4 were similar to the control group values. Differences from the control group were slight and not statistically significant, with the following exception. The anogenital distance relative to the cube root of pup body weight for males in the 250 mg/kg/day group was statistically significantly longer than the control group on PND 4, but was attributed to lower pup body weights for males in this group on PND 4.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Areolae/nipple anlagen retention in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. There were no retained nipples in any group.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Mean pup thyroid weights were unaffected by test substance-administration. Differences from the control group were not statistically significant. Lower mean thyroid/parathyroid weights were noted for female pups in the 50, 100, and 250 mg/kg/day groups, but similar effects were not observed in male pups on PND 13 and there were no correlating effects on thyroid hormone values. In addition, the differences were partially attributed to a high control group value resulting from 1 female that had a thyroid weight of 0.0061 g (vs. 0.0030 g to 0.0037 g for all other control females).
Gross pathological findings:
no effects observed
Description (incidence and severity):
No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead or euthanized in extremis or pups euthanized on PND 13.
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- Thyroid Hormone Analysis (PND 13):
There were no test substance-related effects on thyroid hormone values in the F1 males and females at any dose 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.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 259 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test-item related adverse effects observed up to the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Under the conditions of this screening study, in the absence of any effect on reproductive parameters or any evidence of adverse toxicity for F0 males and females, a dose level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity and F0 systemic toxicity when Javanol was administered orally by gavage to Crl:WI(Han) rats.
Despite lower mean pup body weights and body weight gains at 250 mg/kg/day, there was no clear evidence of an adverse effect on overall animal health; therefore, the NOAEL for F1 neonatal toxicity was considered to be 250 mg/kg/day.
Executive summary:

The objective of this GLP OECD 421 study was to provide preliminary information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through day 13 of postnatal life.

Animals (10 males and 10 females per dose) were administered via oral gavage once daily 0, 50, 100 or 250 mg/kg bw/d. 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 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, thyroid hormones, gross necropsy findings, organ weights, and histopathologic examinations.

There were no test substance-related effects on survival for F0 males and females at any dose level. In the control and 50 mg/kg/day groups, 1 male and 1 female, respectively, were found deadonStudy Day 10. Both animals were noted with macroscopic findings of an esophageal perforation and/or clear fluid accumulation in the thoracic cavity, indicative of dosing errors. All other males and females in the control, 50, 100, and 250 mg/kg/day groups survived to the scheduled necropsy. There were no test substance-related clinical observations at the daily examinations or approximately 1 hour following dose administration for males and females at any dose level.

For F0 males, test substance-related lower mean body weights gains were noted in the 100 and 250 mg/kg/day groups generally throughout the study (Study Days 0–28; statistically significant) with correspondingly lower mean food consumption in the 250 mg/kg/day group during the premating period. These decrements were not considered adverse as they had only minimal impact on mean absolute body weights at 250 mg/kg/day (6.9% lower than controls on Study Day 28; not statistically significant) and mean absolute body weights in the 100 mg/kg/day were comparable to the control group throughout the study. Mean food consumption in the 100 mg/kg/day group males and mean absolute body weights, body weight gains, and food consumption in the 50 mg/kg/day group males were unaffected by test substance administration.

For F0 females, mean body weights and body weight gains in the 250 mg/kg/day group were comparable to the control group during the premating period. Test substance-related lower mean maternal body weight gains with correspondingly lower mean food consumption were noted in this group generally throughout gestation, which resulted in a mean absolute body weight that was slightly lower (4.7%; not statistically significant) than the control group on Gestation Day 20.Due to the minimal magnitude of the effect on absolute body weights, the lower mean body weight gains and reduced food consumption at 250 mg/kg/day during gestation were not considered adverse. During lactation, mean body weights, body weight gains, and food consumption in the 250 mg/kg/day group were unaffected by test substance administration. Mean body weights, body weight gains, and food consumption throughout the treatment period, including during gestation and lactation, were unaffected by test substance administration for females at 50 and 100 mg/kg/day.

No test substance-related effects were noted for estrous cyclicity, reproductive performance (mating, fertility, and pregnancy indices and precoital interval) or delivery observations (including gestation length, gestation index, and implantation sites) at any dose level.

Lower (statistically significant) mean T4levels were noted in the 100 and 250 mg/kg/day group F0males (18.4% and 25.6%, respectively) compared to the control group and were accompanied by higher mean thyroid/parathyroid weights. However, there were no corresponding microscopic correlates, and these changes may be in response to enhanced clearance following liver enzyme induction, reflected by the higher liver weights at 250 mg/kg/day. Given this potential mode of action, and the lack of histopathological findings in the thyroid at 250 mg/kg/day or other evidence of endocrine-disruption, the lower mean T4 concentrations at 100 and 250 mg/kg/day were not considered adverse. There were no test substance-related effects on T4 concentrations for F0 males at 50 mg/kg/day.

Test substance-related, higher mean liver and thyroid/parathyroid weights and lower mean prostate weights for males and lower mean ovary/oviduct weights for females were noted in the 250 mg/kg/day group.[MC1] In the absence of any effects on reproductive performance, and/or microscopic correlates for any of these organ weight changes, these organ weight changes were not considered adverse. Test substance-related, lower mean heart weights were also noted in the 100 mg/kg/day group females and 250 mg/kg/day group males and females; these changes were considered nonadverse, as no microscopic findings in the heart were reported previously at higher dosage levels (Braun et al., 2000).

There were no test substance-related effects on gross necropsy findings and histopathologic findings at any dose level.

The mean numbers of F1 pups born (including number of live newborn pups), percentage of males at birth, postnatal survival (including live birth, viability, and survival indices), clinical condition of the pups, anogenital distance, and areola/nipple retention (males) in the 50, 100, and 250 mg/kg/day groups were unaffected by test substance administration.

Slightly lower (not statistically significant) mean pup birth weights (PND 1) were noted for males and females in the 250 mg/kg/day group compared to the control group. Lower mean pup body weight gains were noted in this group during PND 1–10, with statistical significance achieved during PND 4–10 (males) and 7–10 (females). Increasingly lower mean pup body weights were noted for males and females at 250 mg/kg/day from PND 4 through PND 10, but changes were not statistically significant and the values on PND 4 and 7 were within Charles River Ashland historical control data ranges. During PND 10–13, mean body weight gains at 250 mg/kg/day were comparable to the control group, and mean absolute body weights were approximately 10.6% lower (not statistically significant) than controls on PND 13. Given the evidence of recovery during the last interval prior to euthanasia on PND 13, and the absence of any other evidence of developmental toxicity, the adversity of the changes in pup body weights at 250 mg/kg/day is unclear. There were no test substance-related effects on pup body weights and body weight gains at 50 and 100 mg/kg/day.

There were no test substance-related macroscopic findings noted in F1 pups that were found dead or at the scheduled necropsy on PND 13. There were no test substance-related effects on serum T4 levels or thyroid/parathyroid weights in the F1 pups at any dose level on PND 13.

Under the conditions of this screening study, in the absence of any effect on reproductive parameters or any evidence of adverse toxicity for F0 males and females, a dose level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity and F0 systemic toxicitywhen Javanol was administered orally by gavage to Crl:WI(Han) rats.

Despite lower mean pup body weights and body weight gains at 250 mg/kg/day, there was no clear evidence of an adverse effect on overall animal health; therefore, the NOAEL for F1 neonatal toxicity was considered to be 250 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and OECD Guideline study
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 421, GLP Study (2021):

The objective of this GLP OECD 421 study was to provide preliminary information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through day 13 of postnatal life.

Animals (10 males and 10 females per dose) were administered via oral gavage once daily 0, 50, 100 or 250 mg/kg bw/d. 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 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, thyroid hormones, gross necropsy findings, organ weights, and histopathologic examinations.

There were no test substance-related effects on survival for F0 males and females at any dose level. In the control and 50 mg/kg/day groups, 1 male and 1 female, respectively, were found deadonStudy Day 10. Both animals were noted with macroscopic findings of an esophageal perforation and/or clear fluid accumulation in the thoracic cavity, indicative of dosing errors. All other males and females in the control, 50, 100, and 250 mg/kg/day groups survived to the scheduled necropsy. There were no test substance-related clinical observations at the daily examinations or approximately 1 hour following dose administration for males and females at any dose level.

For F0 males, test substance-related lower mean body weights gains were noted in the 100 and 250 mg/kg/day groups generally throughout the study (Study Days 0–28; statistically significant) with correspondingly lower mean food consumption in the 250 mg/kg/day group during the premating period. These decrements were not considered adverse as they had only minimal impact on mean absolute body weights at 250 mg/kg/day (6.9% lower than controls on Study Day 28; not statistically significant) and mean absolute body weights in the 100 mg/kg/day were comparable to the control group throughout the study. Mean food consumption in the 100 mg/kg/day group males and mean absolute body weights, body weight gains, and food consumption in the 50 mg/kg/day group males were unaffected by test substance administration.

For F0 females, mean body weights and body weight gains in the 250 mg/kg/day group were comparable to the control group during the premating period. Test substance-related lower mean maternal body weight gains with correspondingly lower mean food consumption were noted in this group generally throughout gestation, which resulted in a mean absolute body weight that was slightly lower (4.7%; not statistically significant) than the control group on Gestation Day 20.Due to the minimal magnitude of the effect on absolute body weights, the lower mean body weight gains and reduced food consumption at 250 mg/kg/day during gestation were not considered adverse. During lactation, mean body weights, body weight gains, and food consumption in the 250 mg/kg/day group were unaffected by test substance administration. Mean body weights, body weight gains, and food consumption throughout the treatment period, including during gestation and lactation, were unaffected by test substance administration for females at 50 and 100 mg/kg/day.

No test substance-related effects were noted for estrous cyclicity, reproductive performance (mating, fertility, and pregnancy indices and precoital interval) or delivery observations (including gestation length, gestation index, and implantation sites) at any dose level.

Lower (statistically significant) mean T4levels were noted in the 100 and 250 mg/kg/day group F0males (18.4% and 25.6%, respectively) compared to the control group and were accompanied by higher mean thyroid/parathyroid weights. However, there were no corresponding microscopic correlates, and these changes may be in response to enhanced clearance following liver enzyme induction, reflected by the higher liver weights at 250 mg/kg/day. Given this potential mode of action, and the lack of histopathological findings in the thyroid at 250 mg/kg/day or other evidence of endocrine-disruption, the lower mean T4 concentrations at 100 and 250 mg/kg/day were not considered adverse. There were no test substance-related effects on T4 concentrations for F0 males at 50 mg/kg/day.

Test substance-related, higher mean liver and thyroid/parathyroid weights and lower mean prostate weights for males and lower mean ovary/oviduct weights for females were noted in the 250 mg/kg/day group.[MC1] In the absence of any effects on reproductive performance, and/or microscopic correlates for any of these organ weight changes, these organ weight changes were not considered adverse. Test substance-related, lower mean heart weights were also noted in the 100 mg/kg/day group females and 250 mg/kg/day group males and females; these changes were considered nonadverse, as no microscopic findings in the heart were reported previously at higher dosage levels (Braun et al., 2000).

There were no test substance-related effects on gross necropsy findings and histopathologic findings at any dose level.

The mean numbers of F1 pups born (including number of live newborn pups), percentage of males at birth, postnatal survival (including live birth, viability, and survival indices), clinical condition of the pups, anogenital distance, and areola/nipple retention (males) in the 50, 100, and 250 mg/kg/day groups were unaffected by test substance administration.

Slightly lower (not statistically significant) mean pup birth weights (PND 1) were noted for males and females in the 250 mg/kg/day group compared to the control group. Lower mean pup body weight gains were noted in this group during PND 1–10, with statistical significance achieved during PND 4–10 (males) and 7–10 (females). Increasingly lower mean pup body weights were noted for males and females at 250 mg/kg/day from PND 4 through PND 10, but changes were not statistically significant and the values on PND 4 and 7 were within Charles River Ashland historical control data ranges. During PND 10–13, mean body weight gains at 250 mg/kg/day were comparable to the control group, and mean absolute body weights were approximately 10.6% lower (not statistically significant) than controls on PND 13. Given the evidence of recovery during the last interval prior to euthanasia on PND 13, and the absence of any other evidence of developmental toxicity, the adversity of the changes in pup body weights at 250 mg/kg/day is unclear. There were no test substance-related effects on pup body weights and body weight gains at 50 and 100 mg/kg/day.

There were no test substance-related macroscopic findings noted in F1 pups that were found dead or at the scheduled necropsy on PND 13. There were no test substance-related effects on serum T4 levels or thyroid/parathyroid weights in the F1 pups at any dose level on PND 13.

Under the conditions of this screening study, in the absence of any effect on reproductive parameters or any evidence of adverse toxicity for F0males and females, a dose level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0reproductive toxicity and F0systemic toxicitywhen Javanol was administered orally by gavage to Crl:WI(Han) rats.

Despite lower mean pup body weights and body weight gains at 250 mg/kg/day, there was no clear evidence of an adverse effect on overall animal health; therefore, the NOAEL for F1neonatal toxicity was considered to be 250 mg/kg/day.

Effects on developmental toxicity

Description of key information

No developmental study avalaible on the substance.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The objective of this GLP OECD 421 study was to provide preliminary information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through day 13 of postnatal life.

Oral (gavage) administration of Javanol resulted in test substance-related alterations in body weights, food consumption, thyroid hormones, and organ weights. None of these changes were considered adverse. There were no test substance‑related effects on F0 survival, clinical observations, reproductive performance, gross findings, or histopathology.

Justification for classification or non-classification

Based on the GLP OECD 421 study performed on Javanol, no classification for reproductive or developmental toxicity is necessary according to the (EC) No 1272/2008 Regulation (CLP).

Additional information