Alcohols, C9-11-branched

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation: October 29, 2021
Completion of in-life: July 22, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

yes

Remarks:

Please refer to "Principles of method of other that guideline".

Principles of method if other than guideline:

DEVIATIONS
All deviations that occurred during the study have been authorized/acknowledged by the Study Director, assessed for impact, and documented in the study records. All study protocol deviations that could have impacted the quality or integrity of the study are listed below.
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Formulations and Dosing:
- Protocol Section 9.2.1. states that Group 1 (control group) will receive a dose level of 0 mg/kg/day and that Group 2 will receive a dose level of 100 mg/kg/day. On PND 64, F1 Female No. 4387-11 in the control group was dosed with the 100 mg/kg/day formulation, while Female No. 4382-12 in the 100 mg/kg/day group was dosed with the control group formulation. Female No. 4382-12 was subsequently administered the appropriate dose volume of the 100 mg/kg/day formulation.
- Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because administration of the low-dose formulation to a control group animals on a single day did not affect the clinical condition of the animal; there were no test substance-related effects noted at 100 mg/kg/day. In addition, the female in the 100 mg/kg/day group received the appropriate dose later in the day.

Husbandry:
- Protocol Section 7. states that body weight ranges at initiation of dosing will be 175 to 350 g for males and 125 to 250 g for females. At the initiation of dosing, 17, 26, 17, and 15 F0 males in the control, 100, 300, and 1000 mg/kg/day groups, respectively, weighed 129 to 174 g and 1 F0 female each in the control, 100, and 300 mg/kg/day groups weighed 118 to 124 g.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the animals were the appropriate age and were assigned to groups so that similar mean body weights were achieved.
- Protocol Section 8.3. states that the temperature in the animal room will be 68°F to 77°F (20°C to 25°C). On 02 Nov 2021 and 03 Nov 2021 (acclimation period), The mean daily temperatures in the animal room were 65.7°F and 66.1°F, respectively.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the slight temperature excursions were limited to the acclimation period and had no impact on animal health.
- Protocol Section 8.5. states that samples (approximately 50 g each) will be collected from each lot of feed used on the study and stored frozen (target of -20°C0 for possible additional future phytoestrogen analysis. There was no documentation that feed samples were collected throughout the study.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because analysis of feed samples is not needed for this study.

In-life Observations, Measurements, and Evaluations:
- Protocol Section 10.2. states that cage-side observations will be recorded 1–2 hour(s) following each dose administration. On Study Day 110, the postdosing observation for F0 Male No. 4284 in the 1000 mg/kg/day group was not recorded until 1 hours 22 minutes following dose administration.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the time excursion was minimal and had no impact on data interpretation.
- Protocol Section 10.2. states that cage-side observations will be recorded daily, prior to dosing (on days of scheduled dosing), and at 1–2 hour(s) following each dose administration. On 09 Feb 2022 (Study Day 92), the postdosing observations for 4 F0 females in the control group were not collected until 2 hours 1 minutes following dosing. On 27 Feb 2022 (Study Day 110), the postdosing observation for F0 Male No. 4284 in the 1000 mg/kg/day group was not collected until 2 hours 22 minutes following dosing. On 24 Mar 2022 (PND 41–44), the postdosing observations for 14 and 7 F1 females in the 300 and 1000 mg/kg/day groups, respectively, were not collected until 2 hours 1 minute to 2 hours 8 minutes following dosing. On 16 Apr 2022 (PND 64), the postdosing observation for 1 F1 female in the 100 mg/kg/day group was not collected until 3 hours 36 minutes following dosing. On 22 Apr 2022 (PND 68–72), the postdosing observations for multiple F1 males and females in each group were not collected until 2 hours 1 minute to 2 hours 24 minutes following dosing. On 07 Jun 2022 (PND 116–118), the postdosing observations for 12 F1 males in the 1000 mg/kg/day group were not collected until 2 hours 6 minutes to 2 hours 8 minutes following dosing.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because observations were generally performed minimally outside of the protocol-specified time point. Additionally, no adverse clinical observations were noted on days when observations were collected within the protocol-specified range. Therefore, there was no impact on data interpretation.
- Protocol Section 10.8. states that females will be observed for dystocia (prolonged or difficult labor), or other difficulties and any findings will be recorded. On Postmating Day 22, F0 Female Nos. 4384 and 4455 in the control group, No. 4381 in the 300 mg/kg/day group, and No. 4462 in the 1000 mg/kg/day group were not observed at the morning parturition observation.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the females were observed at the afternoon parturition observation that day; in addition, all of these females were nongravid.
- Protocol Section 10.8.1. states that to reduce variability among the litters, 10 pups from each litter, of equal sex distribution (if possible), will be randomly selected on PND 4. An extra male pup was culled from F2 Litter No. 4437-07 in the control group, resulting in 4 males and 6 females to continue for Cohort 1B evaluations.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because only a single litter was impacted, and a sufficient number of pups were available for F2 assessments.
- Protocol Section 10.9. states that following weaning, twice daily checks for mortality and moribundity will be conducted as described for parental (F0) animals. There was no documentation of a viability check for F1 Litter No. 4452-10 in the 100 mg/kg/day group on PND 20.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because all pups were alive and had no findings at the next scheduled observation.
- Protocol Section 10.9. states that following weaning, detailed clinical and cage-side observations for F1 animals will be conducted as described for parental (F0) animals. Detailed clinical observations were conducted weekly, and cage-side observations were recorded prior to dosing on the days that detail clinical observations were not recorded. On 29 days during the F1 generation, cage-side observations were not recorded for 1–12 animals in the control, 100, 300, and/or 1000 mg/kg/day groups.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because no adverse clinical observations were noted during any cage-side observations on this study; therefore, there was no impact on study interpretation.

Laboratory Evaluations:
- Protocol Section 11.1. states that blood samples for thyroid hormone assessments will be collected prior to noon on each day of collection. On 15 Feb 2022, the thyroid hormone blood sample for F0 Female No. 4382 in the 100 mg/kg/day group was collected at 1204 hours.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the collection time was minimally beyond the protocol-specified time frame.
- Protocol Section 11.4. states that the splenic cells will be used for enumeration of live cell concentration by Nucleoview NC-200 cell counter. However, a Luna-FL Automated Cell Counter was used.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the Lune-FL cell counter has similar capabilities as the Nucleoview NC-200 cell counter.

Postmortem and Pathology:
- Protocol Section 10.13.1. states that pups found dead and any pups that are euthanized in extremis will be dissected (including the heart and brain examined by a mid-coronal slice) by a technique described by Stuckhardt and Poppe, which includes internal verification of sex. Protocol Section 10.9. states that pups will be sexed on Postnatal Day 0, 4, 14, and 21. F1 Pup Nos. 4435-01, 4435-03, 4435-04, 4435-05, and 4435-06 in the 1000 mg/kg/day group were found dead on PND 0 and were not sexed prior to sending for necropsy. The sex of these pups were not documented at internal examination.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because a single litter was affected, and sufficient litter sex distribution data were collected for meaningful data interpretation.
- Protocol Section 10.13.1. states that pups found dead and any pups that are euthanized in extremis will be dissected (including the heart and brain examined by a mid-coronal slice) by a technique described by Stuckhardt and Poppe. A finding of ‘milk not present’ in the stomach was recorded for F2 Pup Nos.4416-14-01 and 4416-14-02 in the control group; however, there was no documentation that any further examination was conducted.
Impact Assessment: This minor documentation error for these 2 control group pups had no impact on the outcome of the study or data interpretation.
- Protocol Section 11.1.2. states that blood samples from F1 pups on PND 21 will be collected via cardiac puncture under isoflurane anesthesia and that following blood collection, pups will be euthanized by exsanguination (bilateral thoracotomy, if necessary). On 02 Mar 2022, there was no documentation of the method of euthanasia for the F1 PND 21 pups following blood collection.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because isoflurane anesthesia is necessary for cardiac puncture blood collection; therefore, there is no impact for omission of this documentation.
- Protocol Section 11.3. states that the left cauda epididymis will be homogenized and evaluated for sperm numbers. Due to erroneous cauda epididymis weights, sperm concentration could not be calculated for F0 Male Nos. 4299 and 4344 in the 300 mg/kg/day group.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because sufficient data were collected in this group to provide meaningful interpretation of epididymal concentration data.
- Protocol Section 11.3. states that the left cauda epididymis will be homogenized and evaluated for sperm numbers. The caput epididymis from F0 Male No. 4277 in the 1000 mg/kg/day group was retained frozen instead of the cauda epididymis, which was placed in modified Davidson’s solution. Therefore, a sperm concentration sample was not analyzed for this male.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because only a single concentration sample was omitted.
- Protocol Section 11.4. states that immediately following euthanasia by carbon dioxide inhalation of the selected animals, the spleen from F1 males and females in Cohort 1A will be harvested, weighed in total and cut in half. One half will be weighed and placed into chilled Hank’s Balanced Salt Solution (HBSS) with 2% fetal bovine serum (FBS). Spleen samples from 3, 2, 1, and 1 Cohort 1A animals in the control, 100, 300, and 1000 mg/kg/day groups, respectively, were each placed in empty tubes at the time of collection. HBSS was not added until 1–23 minutes following collection.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the time of addition of HBSS after the time of collection was minimal and had no impact on analysis or interpretation.
- Protocol Section 12.1. states that culled pups on PND 4 will be anesthetized by isoflurane inhalation (if blood is being collected) or euthanized by an intraperitoneal injection ofsodium pentobarbital. On 19 Jun 2022, there was no documentation of the method of euthanasia for the F2 culled pups that did not have blood samples collected.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because necropsies were conducted indicating that euthanasia was performed according to Testing Facility SOPs.
- Protocol Section 12.2. states that following blood collection, nonselected F1 pups and F2 pups on Postnatal Day 21 will be euthanized by exsanguination (bilateral thoracotomy, if necessary) and subjected to a gross necropsy. At the time of necropsy, tissues and organs will be collected from 10 pups/sex/group (same animals used for blood collection) and placed in 10% neutral buffered formalin. F2 Male No. 4391N05 in the 100 mg/kg/day group was scheduled to have thyroid hormone blood samples and tissues collected. This animal was euthanized by carbon dioxide inhalation. No blood samples were collected; however, the appropriate tissues were collected.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because a single blood sample was omitted; sufficient data were collected from this group to provide for meaningful interpretation of the data.
- Protocol Section 12.2.1. lists the organs to be weighed from PND 21 pups. The liver weight from Male No. 4418L02 and the thymus gland weight from Male No. 4407N04 in the 1000 mg/kg/day group were deleted due to an obvious weighing error. These tissues were reweighed following fixative, but not included in the calculation of means.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because only 1 organ for each animal was affected; sufficient data were collected in this group to provide for meaningful interpretation of organ weight data.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because only a single concentration sample was omitted.
- Protocol Section 11.4. states that immediately following euthanasia by carbon dioxide inhalation of the selected animals, the spleen from F1 males and females in Cohort 1A will be harvested, weighed in total and cut in half. One half will be weighed and placed into chilled Hank’s Balanced Salt Solution (HBSS) with 2% fetal bovine serum (FBS). Spleen samples from 3, 2, 1, and 1 Cohort 1A animals in the control, 100, 300, and 1000 mg/kg/day groups, respectively, were each placed in empty tubes at the time of collection. HBSS was not added until 1–23 minutes following collection.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the time of addition of HBSS after the time of collection was minimal and had no impact on analysis or interpretation.
- Protocol Section 12.1. states that culled pups on PND 4 will be anesthetized by isoflurane inhalation (if blood is being collected) or euthanized by an intraperitoneal injection of sodium pentobarbital. On 19 Jun 2022, there was no documentation of the method of euthanasia for the F2 culled pups that did not have blood samples collected.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because necropsies were conducted indicating that euthanasia was performed according to Testing Facility SOPs.
- Protocol Section 12.2. states that following blood collection, nonselected F1 pups and F2 pups on Postnatal Day 21 will be euthanized by exsanguination (bilateral thoracotomy, if necessary) and subjected to a gross necropsy. At the time of necropsy, tissues and organs will be collected from 10 pups/sex/group (same animals used for blood collection) and placed in 10% neutral buffered formalin. F2 Male No. 4391N05 in the 100 mg/kg/day group was scheduled to have thyroid hormone blood samples and tissues collected. This animal was euthanized by carbon dioxide inhalation. No blood samples were collected; however, the appropriate tissues were collected.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because a single blood sample was omitted; sufficient data were collected from this group to provide for meaningful interpretation of the data.
- Protocol Section 12.2.1. lists the organs to be weighed from PND 21 pups. The liver weight from Male No. 4418L02 and the thymus gland weight from Male No. 4407N04 in the 1000 mg/kg/day group were deleted due to an obvious weighing error. These tissues were reweighed following fixative, but not included in the calculation of means.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because only 1 organ for each animal was affected; sufficient data were collected in this group to provide for meaningful interpretation of organ weight data.
- Protocol Section 12.4. states that for F0 and F1 Cohort 1A animals at the scheduled necropsy, following organ weights and sample retention in 10% neutral buffered formalin, an additional section of the left lobe of liver will be weighed, flash frozen in liquid nitrogen, and stored in a freezer set to maintain -70°C for possible future analysis. The liver samples from F1 Female No. 4433-08 in the 300 mg/kg/day group and No. 4429-12 in the 1000 mg/kg/daygroup were not weighed prior to freezing.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because a single sample from these groups for possible future analysis was impacted.
- Protocol Section 12.7. states that testes will be weighed (separately). An erroneous weight was recorded for the right testis from F0 Male No. 4272 in the 1000 mg/kg/day group; this value was deleted.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because the weighing error was limited a single animal and tissue. Sufficient data were collected for this tissue and group to provide for meaningfulinterpretation.
- Protocol Section 12.7. states that the thymus and adrenal glands from F0 animals will be weighed (fresh). Due to an obvious weighing error, the adrenal glands from F0 Male No. 4342 in the control group, the adrenal and thymus glands from F0 Male No. 4305 in the 100 mg/kg/day group, and the thymus gland from F0 Male Nos. 4276 and 4335 in the 1000 mg/kg/day group were weighed following fixation. In addition, the adrenal and thymus glands, brain, heart, kidneys, liver, ovaries, pituitary, spleen, and uterus (with oviducts and cervix) from F0 Female No. 4393 in the 1000 mg/kg/day group were placed in 10% neutral buffered formalin for approximately 1 minute prior to weight collection. These organs were rinsed in 0.9% saline and weighed. The spleen from F1 Female No. 4423-13 in the control group and mandibular lymph nodes from F1 Male No. 4438-10 in the 300 mg/kg/day group (both Cohort 1A) were weighed after being placed in fixative and were weighed on the same day. These weights were excluded from tabulation.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because sufficient organ weight data were collected in each group to provide for meaningful data interpretation.
- Protocol Section 12.8.1. states that brain lengths and widths will be recorded without knowledge of group assignment. The group assignments were known for all Cohort 2B animals at the brain measurements collection.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because knowledge of group assignment did not affect the measurements of the fixed brains, and therefore did not affect data interpretation.
- Protocol Section 12.8.1. states that the whole brains of PND 22 perfusion pups shall be removed, weighed (including olfactory bulbs) and the size (length [excluding olfactory bulbs] and width) recorded and preserved in 10% neutral buffered formalin (NBF). F1 Cohort 1B Female No. 4380-12 in the control group was necropsied on 05 Mar 2022. At the time of processing tissues on 10 Mar 2022, it was discovered that the head from this female was placed in a jar with no NBF. The head was placed in NBF immediately and allowed to fix for at least 36 hours. The brain was then weighed and measured; these data were excluded from tabulation.
Impact Assessment: This deviation did not negatively impact the quality or integrity of the data or the outcome of the study because only a single brain from the control group was affected, which had no impact on data interpretation.

GLP compliance:

yes

Limit test:

no

Justification for study design:

Justification for animal use:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models that do not use live animals currently do not exist.
The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals: Guideline 443, Extended One-Generation Reproductive Toxicity Study (Jun 2018), which recommends including a sufficient number of mating pairs to yield at least 20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality in each generation of the study, 25 F0 animals/sex/group was an appropriate number of animals to achieve the desired number of animals in each generation.

Justification for route of administration:
The route of administration was oral (gavage) because this is a potential route of exposure to humans.

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C9-11-branched

EC Number:

271-360-6

EC Name:

Alcohols, C9-11-branched

Cas Number:

68551-08-6

Molecular formula:

CnH2n+1OH (n = 10 to 12)

IUPAC Name:

Branched alcohols, C10-12, C11 rich

Test material form:

liquid

Remarks:

clear, colorless

Details on test material:

Batch/Lot No.: 00712B1408
Expiration/Retest Date: 23 Sep 2021
Purity: 99.9%
Storage Conditions: 18°C to 24°C

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were approximately 6 weeks old and weighed between 129 and 212 g for males and between 118 and 193 g for females at the initiation of dosing.
After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing.

Housing:
In groups of 2–3 animals per cage following receipt and throughout the acclimation and premating periods; single/individual following positive signs of mating.
All offspring selected to constitute the F1 generation were group housed (2–3 animals of the same sex and same dosing group together) beginning at weaning and remained in these cages until euthanasia. All offspring selected to constitute Cohort 2B were single-housed overnight, prior to euthanasia on PND 22.
For Cohort 1B, during the breeding period and following successful mating, the F1 males and females and F2 offspring were housed as previously described for the F0 males and females and F1 offspring.

Water:
Municipal tap water, treated by reverse osmosis and ultraviolet irradiation (Ad libitum).

Diet: Ad libitum

Caging:
Solid-bottom cages containing appropriate bedding material (Bed-O-Cobs® or other suitable material).

Cage Identification:
Individual (color-coded) cage cards were affixed to each cage and displayed at least the animal number(s), cage number, group number, dose level, study number, and sex of the animal.

Animals were separated during designated procedures/activities. Where possible, control group animals were housed on a separate rack from the test substance-treated animals.

Environmental Conditions:
The targeted conditions for animal room environment were as follows:
Temperature: 68°F to 77°F (20°C to 25°C)
Humidity: 30% to 70%
Light Cycle: 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions (containing the vehicle and test substance) were prepared approximately weekly and stored between 18°C to 24°C.

Details on mating procedure:

F0 generation:
The F0 animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. 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.

F1 generation:
The F1 animals in Cohort 1B were paired on a 1:1 basis within each treatment group on the first PND 104 (following a minimum of 14 days of vaginal lavage for females). Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please refer to "Any other information on materials and methods incl. tables" below.

Duration of treatment / exposure:

F0 males were dosed orally by gavage for a minimum of 70 consecutive days prior to mating and continuing through the day prior to euthanasia (for a minimum of 17 weeks). F0 females were dosed orally by gavage for a minimum of 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia. The offspring selected for the F1 generation began dosing following weaning until the day prior to euthanasia on PND 52 (Cohort 1 Surplus), PND 91 (Cohort 1A), PND 78 (Cohort 2A), and following the reproductive assessment (Cohort 1B); pups in Cohort 2B were not directly dosed prior to euthanasia on PND 22.

Frequency of treatment:

Daily

Details on study schedule:

F0 animals were assigned randomly into groups (males and females were randomized separately).
To reduce variability among the F1 litters, 10 pups/litter, 5 pups/sex when possible, were randomly selected on PND 4. For the F1 generation, 3 F1 pups/sex/litter from all litters were randomly selected prior to weaning and assigned to the different cohorts. Animals assigned to Cohort 1B were maintained on study for breeding when the animals were between 91 and 104 days old to generate F2 generation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
The dose levels were selected based on information provided by the Sponsor. The high dose (1000 mg/kg/day) was chosen based on lack of F0 toxicity findings from an extended OECD 422 screening study and 90-day data on adult males and females.

Fasting period before blood sampling for clinical biochemistry:
All animals will (including those not scheduled for clinical pathology assessments) will be fasted overnight prior to blood collection.

Examinations

Parental animals: Observations and examinations:

Observations:
Detailed clinical observations were recorded weekly, prior to dosing beginning with the first day of dosing administration (F0 Generation), or prior to dosing, following weaning (F1 generation); animals were removed from the cage. During the dosing period, cage-side observations were recorded daily, prior to dosing (on days of scheduled dosing), and at 1–2 hour(s) following each dose administration. Cage-side observations were not conducted prior to dosing on days that detailed clinical observations were performed. The absence or presence of findings was recorded for individual animals. During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

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.

Body Weight:
Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy (F0 Generation), or weekly following weaning (F1 Generation). Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 0 (when possible) 1, 4, 7, 14, and 21. A fasted weight was recorded on the day of necropsy. Terminal body weights were not collected from animals found dead or euthanized moribund.

Food consumption:
Food consumption was quantitatively measured weekly throughout the study (or following weaning for F1 generation), except during the mating period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 14, and 21.
F0 generation: For females without evidence of mating or those that fail to deliver, food consumption was not recorded following the end of the breeding period or Post mating Day 25, respectively. Food consumption was also not recorded for females with total litter loss, following loss of the litter. Similarly, food consumption was not recorded, following weaning of offspring, for females that deliver (between weaning and euthanasia).

Food Efficiency:
Body weight gained as a percentage of food consumed was calculated and reported.

Parturition:
The day parturition was initiated was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). During the period of expected parturition, females were observed twice daily for initiation and completion of parturition and for dystocia (prolonged or difficult labor) 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. The dam and litter remained together until weaning on PND 21.

Thyroid hormone analysis:
Blood samples for thyroid hormone analyses were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) from the jugular vein into tubes without anticoagulants. For generation F0, this was done on the day of the scheduled necropsy (week 18, 10 animals/sex/group). For generation F1, this was done an PND91. The samples were analyzed for: Triiodothyronine (T3, UHPLC/MS/MS), Thyrosine (Total T4, UHPLC/MS/MS), and Thyroid Stimulating Hormone (TSH, validated Luminex Bead Based (TSH) assay).

Clinical Pathology:
Animals were fasted overnight prior to blood collection. Urine was collected overnight using metabolism cages. Blood samples for hematology and/or clinical chemistry were collected from the jugular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.
K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for clinical chemistry were collected without anticoagulants. In addition, blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped, and retained for possible future evaluation. The samples were taken on the day of scheduled necropsy (Week 18, Generation F0), PND 91 (Generation F1).

The following parameters were tested (Hematology, Coagulation, Clinical Chemistry, Urinalysis:
Hematology:
Total leukocyte count (WBC)
Erythrocyte count (RBC)
Total hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (Platelet)
Absolute reticulocyte count (RETIC Absolute)
Differential leukocyte count -Absolute*
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)
Red cell distribution width (RDW)
Hemoglobin distribution width (HDW)
Platelet estimatea
Red cell morphology (RBC Morphology)*

* If a manual differential was performed, and the manual data were accepted and reported instead of the automated differential data, results included platelet estimates and RBC morphology.

Coagulation:
Blood samples were processed for plasma fore analysis for the following parameters started.
Activated partial thromboplastin time (APTT)
Prothrombin time (PT)
Sample quality*

*Includes degree of hemolysis, lipemia, and icterus (individual tables only).

Clinical Chemistry:
Blood samples were processed for serum fore analysis for the following parameters started.
Albumin
Total protein
Globulin [by calculation]
Albumin/globulin ratio (A/G Ratio) [by calculation]
Total bilirubin (Total BILI)*
Urea nitrogen
Creatinine
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma glutamyltransferase (GGT)
Bile acids
Glucose
Total cholesterol (Cholesterol)
Calcium
Chloride
Phosphorus
Potassium
Sodium
Sorbitol dehydrogenase (SDH)
Triglycerides (Triglyceride)
Appearance**

* When total bilirubin was > 0.5 mg/dL( > 8.55 µmol/L), direct bilirubin was also measured, and indirect bilirubin was calculated.
** Includes degree of hemolysis, lipemia, and icterus; presented on individual other chemistry data tables.

Urinalysis:
Urine samples were processed and analyzed for the following parameters:
Specific gravity (SG)
pH
Urobilinogen (URO)
Total volume (TVOL)
Color (COL)*
Clarity (CLA)*
Protein (PRO)*
Glucose (GLU)*
Ketones (KET)*
Microscopy of sediment
Bilirubin (BIL)*
Occult blood (BLD)*
Leukocytes (LEU)*

* Presented on individual data tables

Oestrous cyclicity (parental animals):

Please refer to "Any other information on materials and results incl. tables".

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.
Litter parameters were defined as follows:

Mean Live Litter Size = (Total Viable Pups on PND 0) / (No. Litters with Viable Pups on PND 0)

Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (% Per Litter) = (Sum of (Viable Pups/Litter on PND 0 or PND 4 [Pre-Selection]/No. of Pups Born/Litter)*) / (No. of Litters/Group) x 100

Postnatal Survival for All Other Intervals (% Per Litter) = (Sum of (Viable Pups/Litter at End of Interval N/Viable Pups/Litter at Start of Interval N)*) / (No. of Litters/Group) x 100

Where N = PND 0–1, 1–4 (Pre-Selection), 4 (Post-Selection)–7, 7–14, 14–21, or 4 (Post-Selection)–21

* = Pups that were found dead or euthanized due to drowning, mechanical injury, inadvertent removal from room, group termination or due to death of the dam were excluded from pup viability calculations.

Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia. Litters that were euthanized prior to scheduled euthanasia due to reasons unrelated to test substance administration (e.g., drowning, mechanical
injury, inadvertent removal from room, early group termination, or death of the dam) were not considered to be a total litter loss on the data tables and were not included in the pup viability calculations.

Observations:
The animals were removed from the cage, and a detailed clinical observation was performed on PND 1, 4, 7, 14, and 21. Any abnormalities in nesting and nursing behavior were recorded.

Sex determinations:
Pups were individually sexed on PND 0 or 1, 4, 14, and 21.

Body weight:
Pups were weighed individually on PND 1, 4, 7, 14, and 21.

Anogenital Distance:
On PND 1, the anogenital distance was measured for all pups and was defined as the distance from the cranial margin of the anus to the caudal margin of the genital tubercle. Absolute and relative values (to cube root of body weight) were reported.

Areolas/Nipple Anlagen Retention:
On PND 13, all male pups were evaluated for the presence of thoracic nipples/areola. The number of nipples was recorded.

Thyroid Hormone Analysis:
Blood samples for thyroid hormone analyses were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) via cardiac puncture of animals anesthetized by inhalation of isoflurane into tubes without anticoagulants.

Samples were taken at PND 4 from culled pups and pooled by litter until a total of 10 samples/dose level were obtained. Samples from the first 10 litters at each level were used. Samples were taken at PND 21 from non-selected pups (10/sex/group) and pooled by litter and sex.
The samples were analyzed for:
Triiodothyronine (T3) (UHPLC/MS/MS)
Thyroxine (Total T4) (UHPLC/MS/MS)
Thyroid Stimulating Hormone (TSH) (validated Luminex Bead Based (TSH) assay)

Balanopreputial Separation:
Each male was observed for balanopreputial separation beginning on PND 35. Examination of the males was continued daily until balanopreputial separation was present, and the age of attainment was recorded. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a persistent thread of tissue between the glans and prepuce was recorded.

Vaginal Patency:
Each female was observed for vaginal perforation beginning on PND 25. Examination of the females was continued daily until vaginal patency was present, and the age of attainment was recorded. Body weights were recorded at the age of attainment of this landmark. In addition, the appearance of a vaginal thread was recorded.

Auditory Startle Response (Cohort 2A):
An auditory startle response test was performed on animals assigned to Cohort 2A on PND 24 (± 1 day). The same animals were tested at each interval. Auditory startle response testing was performed in a room equipped with a white-noise generation system. Each test session consisted of a 5-minute acclimation period with a 68 dB to 72 dB broadband background white noise. The startle stimulus for each trial was a 110 dB to 120 dB mixed-frequency noise burst stimulus, approximately 20 ms in duration. Responses were recorded during the first 100 ms following the onset of the startle stimulus for each trial. Each test session consisted of 50 trials, with 8-seconds between each trial. Startle response data were analyzed in 5 blocks of 10 trials each. Startle response measurements obtained were PEAK (peak response amplitude) and Tpeak (latency to PEAK).

FOB Assessments (Cohort 2A)
FOB findings were recorded for all Cohort 2A animals on PND 65 prior to motor activity assessment. The FOB used is based on previously developed protocols. Testing was performed by the same trained technicians, when possible, who did not know the animal’s group assignment and was performed at approximately the same time each day. The FOB was performed in a sound-attenuated room equipped with a white noise generator. All animals were observed for the following parameters as described below.

Home cage observations:
Posture
Convulsions/tremors
Feces consistency
Biting
Palpebral (eyelid) closure

Handling Observations:
Ease of removal from cage
Lacrimation/chromodacryorrhea
Piloerection
Palpebral closure
Eye prominence
Red/crusty deposits
Ease of handling animal in hand
Salivation
Fur appearance
Respiratory rate/character
Mucous membranes/eye/skin color
Muscle tone

Open Field Observations:
Mobility
Rearing
Convulsions/tremors
Grooming
Bizarre/stereotypic behavior
Time to first step (seconds)
Gait
Arousal
Urination/defecation
Gait score
Backing
Note: Open field observations were evaluated over a 2-minute observation period.

Sensory Observations:
Approach response
Startle response
Pupil response
Forelimb extension
Air righting reflex
Touch response
Tail pinch response
Eyeblink response
Hindlimb extension
Olfactory orientation

Neuromuscular Observations:
Hindlimb extensor strength
Hindlimb foot splay
Grip strength-hind and forelimb
Rotarod performance

Physiological Observations:
Catalepsy
Body temperature
Body weight

Motor activity (Cohort 2A):
Motor activity was assessed on animals assigned to Cohort 2A on PND 65. The same animals were tested at each interval using a series of infrared photobeams to quantify each animal’s motor activity. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator, and black enclosures were used to decrease the potential for distraction. Data were collected in 5-minute epochs over a period of 60 minutes, and the data were reported in 10-minute subintervals. Total motor activity was defined as a combination of
fine motor skills (i.e., grooming; interruption of 1 photobeam) and ambulatory motor activity (e.g., interruption of 2 or more consecutive photobeams).

Postmortem examinations (parental animals):

For a summary overview, please refer to "Any other information on materials and methods incl. tables".

Unscheduled deaths:
A necropsy was conducted for animals that died on study, and specified tissues were saved. If necessary for humane reasons, animals were euthanized. These animals underwent necropsy, and specified tissues were retained. For females found dead or euthanized for humane reasons during gestation, the number and location of viable fetuses, corpora lutea, and (former) implantation sites were recorded. For females found dead or euthanized during lactation, the number of corpora lutea (through Lactation Day 4) and former implantation sites were recorded. Recognizable fetuses were examined externally and discarded (generation F0), or preserved in 10% neutral buffered formalin (generation F1). All pups were euthanized by an intraperitoneal injection of sodium pentobarbital in the scapular region and necropsied.

Scheduled Euthanasia:
All surviving animals, including females that failed to deliver or with total litter loss, were euthanized by carbon dioxide inhalation following the selection of the F1 generation (generation F0).

Cohort 2: All animals were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and perfused in situ with fixative (4% paraformaldehyde solution in 0.1M phosphate buffer).

Sperm Evaluations:
Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid-line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a minimum 10-minute incubation period, a sample of sperm was loaded onto a slide with a 100-µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported.
The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
The left testis and cauda epididymis from all males were weighed and stored frozen. The left cauda epididymis was homogenized and analyzed for determination of homogenization resistant spermatid count. An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20 µm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample.

Necropsy:
All 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. Special attention was paid to the organs of the reproductive system. The numbers of implantation sites and 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. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.

Cohort 2: Brains from all animals on PND 22 (Cohort 2B) and the central and peripheral nervous system tissues from all animals on PND 78 (Cohort 2A) were dissected. Any abnormal coloration or lesions of the external brain and spinal cord were recorded.

Organ Weights:
The organs identified 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 noted. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Organs that were weighted are
Generation F0:
Adrenal glands
Brain
Epididymidesa (total and cauda)
Heart
Kidneys
Liver
Ovaries
Pituitary gland
Prostate gland
Seminal vesicles with coagulating glands
(with accessory fluids)
Spleen
Testes*
Thymus gland
Thyroids with parathyroids**
Uterus with oviducts and cervix

* These paired organs were weighed separately.
** After fixation.

Generation F1 (Cohort 1):
Adrenal glands*
Brain*
Epididymides** (totalc*** and cauda)
Heart*
Kidneys*
Liver*
Lymph nodes – iliac and mandibular****
Ovaries
Pituitary gland
Prostate gland
Seminal vesicles with coagulating glands (with accessory fluids)
Spleen*
Testes**
Thyroids with parathyroids*, *****
Thymus glanda
Uterus with oviducts and cervix

* Collected from F1 Cohort 1A animals only.
** These paired organs were weighed separately.
*** Total weight only for Cohort 1B.
**** Lymph nodes associated with, and distant from, the route of exposure were collected from Cohort 1A only (10 rats/sex/group [1 pup/litter]).
***** After fixation.

Generation F1 (Cohort 2):
The whole brains were removed (including olfactory bulbs), weighed, and the dimensions (length [excluding olfactory bulbs] and width) were recorded without knowledge of treatment group.

Tissue collection and Preservation:
Representative samples of the tissues identified below were collected from all animals and preserved in 10% neutral buffered formalin.

Collected tissues were:
Adrenal glands (2)
Aorta
Bone with marrow (sternebrae)
Brain
Coagulating glands (2)
Eyes with optic nerve (2)*
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Peyer’s Patches
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Lacrimal/Harderian glands
Liver (section of 2 lobes)**
Lungs (including bronchi, fixed by inflation with fixative)
Lymph node (axillary [2], iliac [2], mandibular [2]***, and mesenteric)
Ovaries**** and oviducts (2)
Peripheral nerve (sciatic)***
Pituitary
Prostate
Mandibular salivary gland (2)***
Seminal vesicles (2)
Skeletal muscle (quadriceps)
Skin with mammary gland*****
Spinal cord (cervical)
Spleen
Testes with epididymides (2) and vas deferens (1)******, *******
Thymus
Thyroids with (with parathyroids, if present [2])
Urinary bladder
Uterus with cervix and vagina
All gross lesions (all groups)

* Fixed in Davidson’s solution.
** After organ weight and sample collection in 10% neutral buffered formalin, a section of the left liver lobe was weighed and flash frozen in liquid nitrogen and stored in a freezer set to maintain -70°C for possible analysis.
*** Only 1 examined.
**** Ovaries were fixed in 10% neutral buffered formalin for approximately 48 hours following which all ovaries were transferred to 70% ethanol.
***** For females, a corresponding section of skin was taken from the same anatomic area for males.
****** Testis (right) and epididymis (right and remainder of left) were fixed in modified Davidson’s solution. Both testes and epididymides from animals found dead or euthanized in extremis were fixed in modified Davidson’s solution. Care was taken to ensure separation between the left and right organs.
******* If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis were fixed in modified Davidson’s solution and the right testis and epididymis were homogenized.

Generation F1 (Cohort 2):
Brains from all animals perfused in situ on PND 22 (Cohort 2B) were collected and preserved in 10% neutral buffered formalin (for exceptions, see Appendix 1). In addition, representative samples of the tissues identified below were collected from all animals perfused in situ on PND 78 (Cohort 2A), unless otherwise indicated.

Brain: Olfactory bulbs, cerebral cortex (2 levels), hippocampus/dentate gyrus, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons and medulla oblongata (full coronal section was prepared).
Spinal cord: At cervical swellings C3-C7 & at lumbar swellings T13-L4
Trigeminal ganglia/nerves**
Lumbar dorsal root ganglia at T13-L4*
Lumbar dorsal root fibers at T13-L4*
Lumbar ventral root fibers at T13-L4*
Cervical dorsal root ganglia at C3-C7*
Cervical dorsal root fibers at C3-C7*
Cervical ventral root fibers at C3-C7*
Pituitary gland
Cauda equina nerves + ***, ****
Sciatic nerves + ***
Sural nerves + ***
Tibial nerves + ***
Peroneal nerves + ***
Nasal tissue with olfactory epithelium
Optic nerves**
Eyes**
Skeletal muscle: gastrocnemius.
Other sites: if deemed necessary.

** Both processed and evaluated microscopically
*** Two sections of each peripheral nerve were prepared. The cross section embedded in hard plastic (Spurr’s resin, Epon, methyl methacrylate, etc.); longitudinal section embedded in paraffin.
**** Preserved in situ for possible future examinations.
* Four to 6 tissues were collected at necropsy; 2 tissues were evaluated microscopically.
+ The right nerve was processed for microscopic examination. The tissues from the left hind leg were collected and preserved for possible future evaluation.

Histology:
Generation F0: Tissues identified listed for Tissue collection and Preservation from all animals in the control and high-dose groups and from all animals found dead and euthanized in extremis, as well as gross lesions and target tissues (adrenal glands, kidneys, liver, lungs, and thyroid glands) from all animals in all groups, and reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below.
Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. Testes and epididymides from males that were found dead or euthanized in extremis were stained with H&E only. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
A single section was taken from all F0 females for a qualitative bilateral evaluation of each ovary.

Generation F1 (Cohort 1): Tissues identified listed for Tissue collection and Preservation from F1 animals in Cohort 1A in the control and high-dose groups and from all animals found dead and euthanized in extremis, as well as gross lesions and target organs (adrenal glands [females only], right epididymis, kidneys, liver, lungs, and thyroid glands) from all animals in all groups were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below.
Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. Testes and epididymides from males that were found dead or euthanized in extremis were stained with H&E only. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
Five (5) sections were taken approximately 100 µm apart from the inner third of each ovary from all F1 Cohort 1A females at the scheduled termination. In addition, a single section was taken from all F1 Cohort 1A females for a qualitative bilateral evaluation of each ovary. For females found dead or euthanized in extremis, a single section from each ovary was qualitatively evaluated.
The coagulating glands, ovaries, pituitary gland, prostate gland, seminal vesicles, testes with epididymides, uterus with cervix and vagina, and gross lesions from Cohort 1B animals were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Testes and epididymides were processed as described for Cohort 1A animals.

Generation F1 (Cohort 2): The brains from all animals perfused in situ on PND 22 (Cohort 2B) were embedded in paraffin. For all animals perfused in situ on PND 78 (Cohort 2A), the central nervous system tissues identified in Text Table 40 were embedded in paraffin, and the peripheral nervous system tissues were embedded in plastic.
Other nervous system tissues listed in the table above all groups from Cohorts 2A and 2B were trimmed, processed and embedded in paraffin, except that the grossly trimmed cross-sections of peripheral nerve specimens will be osmicated and embedded in hard plastic (Spurr’s resin, Epon, methyl methacrylate, etc.). Trigeminal nerves and cervical/lumbar root fibers are not considered to be peripheral nerves.
Brains from control and high-dose groups of Cohorts 2A and 2B were microtomed and stained by hematoxylin and eosin (for qualitative histopathological examination) and by Luxol fast blue/Cresyl violet (for evaluation of myelin and/or morphometric analysis). Handling of the brain on all Cohorts 2A and 2B animals was conducted in accordance with the morphometric analysis section. All other nervous system tissue (i.e., those tissues other than brain listed in table above) from control and high-dose groups from Cohort 2A were processed to slides and stained with hematoxylin and eosin for qualitative examination by light microscopy, with the exception of cross-section specimens of peripheral nerves. The cross-section specimens of peripheral nerves in hard plastic were sectioned at 500–700 nm thickness by ultramicrotomy and stained with toluidine blue for evaluation of myelin.

Histopathology:
Generation F0: Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified tissue collection and preservation for microscopic examination were evaluated from all animals in the control and high-dose groups and from all animals found dead and euthanized in extremis. Gross lesions and target tissues were examined from all animals in all groups. In addition, reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, were subjected to a histopathologic evaluation.
Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). Uterine and ovarian histopathology were considered in light of the terminal estrous stage.

Generation F1: Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified listed for Tissue collection and Preservation for microscopic examination were evaluated from all Cohort 1A animals in the control and high-dose groups and from all animals found dead and euthanized in extremis. For all Cohort 1B animals in the control and high-dose groups, reproductive tissues (coagulating glands, prostate, seminal vesicles, testes, epididymides, uterus, ovaries, cervix, vagina) were evaluated. Gross lesions and target organs were examined from all animals in all groups in both cohorts.
In addition, reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, were subjected to a histopathologic evaluation. Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). When possible, sections of the rete testis were examined in the F1 Cohort 1A males.
For all F1 Cohort 1A females in the control and high-dose groups at scheduled termination, a quantitative histopathologic evaluation of multiple sections was conducted. This examination included enumeration of the total number of primordial follicles primordial follicles. Uterine and ovarian histopathology were considered in light of the terminal estrous stage.

Splenic Lymphocyte Immunophenotyping (Cohort 1A):
The spleen from 10 F1 animals/sex/group in Cohort 1A was harvested, weighed, and cut in half. One-half was placed into chilled Hank’s Balanced Buffer Salt solution with 2% fetal bovine serum; the remaining half was placed in 10% neutral buffered formalin.
For the leukocyte subset and phenotypes, please refer to "Any other information an materials and methods incl. tables".
Immunophenotyping results were expressed as relative frequency (% gated) of total spleen leukocytes for each subset. The absolute lymphocyte count (cells/organ) of each subset was calculated by multiplying each leukocyte subset frequency (%) with the total leukocyte count. The following formula was used in generating the absolute number of different subsets of lymphocytes in the spleen:

Absolute number of cells (x10E6 /tissue) = (([(whole organ wt.)*(live cell conc)*(vol of cells)) / (partial organ wt.)) x ((cell subset frequency) / 100) x (0.000001)

F1 Neuropathology (Generation F1, Cohort 1 +2):
Due to the large number of animals to be perfused for neuropathological assessment, perfusions for Cohorts 2A and 2B were performed over several days. The perfusions were performed such that both sexes and all treatment groups were approximately equally represented across each day. The order of perfusions each day was also counterbalanced by sex and treatment group (i.e., 1 male from Group 1, 1 male from Group 2, 1 male from Group 3, 1 male from Group 4, 1 female from Group 1, etc.).
Neuropathological evaluation was performed by a board-certified veterinary pathologist. For animals perfused in situ on PND 22 (Cohort 2B), sections from all major brain regions (including olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain, brainstem, and cerebellum) were evaluated from all animals in the control and high-dose group. For all animals perfused in situ on PND 78 (Cohort 2A), tissues identified in listed for Tissue collection and Preservation for microscopic examination were evaluated from all animals in the control and high-exposure group.

Morphometric Analysis (Generation F1, Cohort 2A):
Histopathological examination included a simple morphometric analysis for Cohort 2A animals on PND 78. Simple linear morphometric measurements were obtained from homologous sections of the brain at 3 levels. The three slides corresponding to these blocks were scanned using a Hamamatsu Nanozoomer whole slide scanner and imported into the Visiopharm software for measurements. Microscopically, up to 11 linear morphometric measurements, were taken in a blinded random fashion from control and high dose group animals.
The following linear measurements were taken on those homologous sections selected by the neuropathologist:
1. Thickness of the frontal cortex bilaterally. This measurement was taken from the dorsal portion of the cerebral cortex within the coronal section passing through the region of the optic chiasm.
2. Thickness of the parietal cortex bilaterally. This measurement was taken from the dorsolateral portion of the cerebral cortex within the coronal section taken through the optic chiasm.
3. Diagonal width (maximum cross-sectional width) of the caudate-putamen bilaterally. This measurement was performed on the coronal section taken at the level of the optic chiasm.
4. Thickness of the corpus callosum bilaterally just lateral to its midpoint at the level of Layer 2 of the overlying cingulate gyrus. This measurement was taken within the section passing through the optic chiasm.
5. Thickness of all hippocampal layers combined, bilaterally, extending from the alveus to just lateral to the inferior blade of the dentate gyrus. This measurement was performed within the section taken at the level of the infundibulum.
6. Maximum height of the cerebellum at the level of the deep cerebellar nuclei, extending from the roof of the fourth ventricle to the dorsal surface. Note that Lobules 1 or 10 of the cerebellum (which protrude into the fourth ventricle) may have been excluded from measurement if inconsistently present on section.

Postmortem examinations (offspring):

For summary please refer to "Any other information on materials and methods incl, tables".

Unscheduled Deaths:
A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary for humane reasons, animals were euthanized. 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. Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead or euthanized for humane reasons after PND 4.
F1 Litter: Pups with external abnormalities that would warrant further skeletal examination were eviscerated and stained for subsequent skeletal evaluation.

Scheduled Euthanasia:
On PND 4, culled pups were euthanized by exsanguination (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital. On PND 21, non-selected pups were euthanized by exsanguination (those pups used for blood/thyroid collection) or carbon dioxide inhalation.

Necropsy:
On PND 4, 1 culled pup/sex/litter was subjected to a complete necropsy examination. Pups were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. All remaining culled pups were discarded without examination.
On PND 21, non-selected pups were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system.

Organ Weights:
The organs identified below were weighed at necropsy from up to 10 non-selected F1 pup/sex/group on PND 21. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Weighed organs:
Brain
Liver
Spleen
Thymus
Thyroid*, **

* After fixation.
** Weighed from same 10 pups/sex/group that were used for thyroid hormone blood collection.

Tissue Collection and Preservation:
Representative specimens with malformations and gross lesions from offspring found dead or euthanized for humane reasons were preserved in 10% neutral buffered formalin.
Representative samples of the tissues identified below were collected from 1 culled pup/sex/litter on PND 4 and preserved in 10% neutral buffered formalin.

Collected tissues:
Trachea (with thyroid gland)
All gross lesions

Representative samples of the tissues identified below were collected from up to 10 non-selected F1 pup/sex/group on PND 21 (the same animals used for blood collection) and preserved in 10% neutral buffered formalin.

Collected tissues:
Brain
Liver
Ovaries
Skin with mammary gland*
Spleen
Testes (2)**
Thymus
Thyroid glands
All gross lesions

* For females; corresponding section of skin was taken from the same anatomical area for males.
** Fixed in modified Davidson’s solution.

Statistics:

Please refer to "Any other information on materials and methods incl. tables".

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Dermal irritation (if dermal study):

not examined

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

not examined

Ophthalmological findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Histopathological findings: neoplastic:

not examined

Effect levels (P0)

Key result

Dose descriptor:

other:

Remarks on result:

other: Effect levels will be provided when the final report is issued (anticipate Aug 2023)

Results: P1 (second parental generation)

General toxicity (P1)

Dermal irritation (if dermal study):

not examined

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

not examined

Ophthalmological findings:

not examined

Histopathological findings: neoplastic:

not examined

Effect levels (P1)

Key result

Dose descriptor:

other:

Remarks on result:

other: Effect levels will be provided when the final report is issued (anticipate Aug 2023)

Results: F1 generation

General toxicity (F1)

Dermal irritation (if dermal study):

not examined

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

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

other:

Remarks on result:

other: Effect levels will be provided when the final report is issued (anticipate Aug 2023)

Results: F2 generation

General toxicity (F2)

Dermal irritation (if dermal study):

not examined

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

Histopathological findings:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

other:

Remarks on result:

other: Effect levels will be provided when the final report is issued (anticipate Aug 2023)

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Treatment related:

not specified

Applicant's summary and conclusion

Conclusions:

The results of the extended one generation study is still under investigation for further assessment. We've received a draft study report and we will be submitting an updated registration when the final report is available for the extended one generation study (anticipated August 2023). Materials /methods and control information are shown in the robust study summary. 

Executive summary:

The objective of this study was to evaluate the potential adverse effects of the test substance, isoundecanol, when given by oral (gavage) administration to rats, on reproduction in an extended one-generation study. This included evaluation of life stages not covered by other types of toxicity studies and test for effects that may occur as a result of pre- and postnatal chemical exposure. 

F0 animals were dosed via oral gavage daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. The offspring in the F1 generation were potentially exposed in utero during gestation. Direct offspring exposure during the  pre-weaning period was demonstrated on a previous dose range-finding OECD 422 study where quantifiable levels of isoundecanol were obtained in maternal milk obtained from dams exposed at a dose level of 750 mg/kg/day. The offspring selected to become the F1  generation were dosed beginning at weaning and continuing until euthanasia (as  appropriate for the relevant cohort).

The following parameters and end points were evaluated in this study: mortality, clinical  signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, pre- and postweaning developmental landmarks, neurobehavior, thyroid hormones, clinical pathology, macroscopic findings, sperm parameters, immunophenotyping, organ weights and measurements, microscopic examinations, and neuropathologic and brain morphometric examinations.

F1 animals were further subdivided into cohorts following weaning and specifically evaluated for the following:

Cohort 1 (A and B) for reproductive/developmental toxicity and Cohort 2 (A and B) for developmental neurotoxicity testing. Animals assigned to Cohort 1A were evaluated on PND 91 (including estrous cycles and sperm evaluations) and Cohort 1B animals were  maintained on study for the assessment of reproductive performance and to generate F2 offspring. Animals assigned to Cohort 2B were evaluated on PND 22 (brain morphometry) and Cohort 2A animals were maintained on study until PND 78 for neurobehavioral testing and for evaluation of adult neuropathology.

 

The results of the extended one generation study is still under investigation for further assessment. We've received a draft study report and we will be submitting an updated registration when the final report is available for the extended one generation study (anticipated August 2023). Materials /methods and control information are shown in the robust study summary.