Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07 Mar 2017 to 07 Jul 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02 Dec 2015 to 22 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this investigative study was to evaluate the short and long-term effects of the test substance on body weight, body weight gain, plasma prolactin, leptin and adiponectin levels, hypothalamic dopaminergic neurons, oestrous cyclicity (including the transition into reproductive senescence), and other key parameters following dietary exposure of female Han Wistar rats for up to 18 months. Females were divided into Cohorts and Subsets for euthanasia and post-mortem activities conducted during Study Weeks 13, 26, 52, 66, and 80. The data collected in this study has been used to test a mode of action hypothesis for uterine tumours in the rat, observed at the top dose in a previously conducted carcinogenicity study with test substance and to compare and contrast with another test substance, a soil and plant metabolite of test substance.

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI[Han]

Sex:

female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

80 weeks

Frequency of treatment:

Continuously

Dose / conc.:

500 ppm

Remarks:

Group 2: Test substance 1. Mean dietary equivalent to 28 mg/kg bw/day

Dose / conc.:

3 000 ppm

Remarks:

Group 3: Test substance 1. Mean dietary equivalent to 194 mg/kg bw/day

Dose / conc.:

3 000 ppm

Remarks:

Group 4: Test substance metabolite. Mean dietary equivalent to 176 mg/kg bw/day

No. of animals per sex per dose:

145 per dose were used, arranged in the following cohorts:
Week 13 and 26 animals: 20 females per dose
Week 52, 66 and 80 animals - Subset 1: 20 animals used for blood and frozen tissue collection, a range of organ weights, and histopathology of the pituitary.
Week 52, 66 and 80 animals - Subset 2: 15 animals used for collection of perfused brain tissue for immunohistochemistry, in situ hybridisation and stereology. In addition, histopathology was performed on a range of fixed tissues.

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily, once in the morning and once in the afternoon, for appearance, behaviour, moribundity, mortality, and signs of overt toxicity. Detailed physical examinations were recorded weekly for all females throughout the study period. In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal; only positive findings were recorded.

BODY WEIGHTS
Individual body weights were recorded once a week, beginning 1 week prior to the start of test diet administration, for the first 26 weeks and biweekly thereafter, until termination. Mean body weights and mean body weight changes were calculated for the corresponding intervals. For naïve male rats, individual body weights were recorded on a biweekly basis for veterinary health monitoring purposes only. These data were not reported and were maintained in the study records.

FOOD CONSUMPTION AND TEST SUBSTANCE CONSUMPTION
Food consumption was recorded once a week, beginning 1 week prior to the start of test diet administration, for the first 26 weeks and biweekly thereafter, until termination. Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. In addition, food efficiency (body weight gained as percent of feed consumed) was calculated. When food consumption could not be determined for a given interval (due to spillage, termination of the social group, weighing error, obvious erroneous value, selection for necropsy, etc.) group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable were left blank or designated as “NA” on the individual report tables. The mean amounts of test substance consumed (mg/kg/day) per dose group were calculated by multiplying the concentration of the test substance in the diet (mg/kg of diet) by the g/animal/day food consumption value and dividing by the body weight (in kg) for each animal.

OESTROUS CYCLES
Daily vaginal lavages were performed for all surviving females during Weeks 3 - 4, 7 - 8, 11 - 12, 16 - 17, 20 - 21, 24 - 25, 29 - 30 - 31, 34 - 35, 38 - 39, 42 - 43, 46 - 47, 50 - 51, 55 - 56 - 57, 60 - 61, 64 - 65, 68 - 69, 73 - 74, and 77 - 78 - 79 of the study to determine the stage of the oestrous cycle. For individual females scheduled for necropsy at the end of any of the selected assessment periods (for a given cohort), daily vaginal lavages continued to be performed up to and including the day of scheduled blood sampling/termination. Beginning with Weeks 46 - 47 and continuing for the remainder of the study, the level of mucus present in wet lavage specimens was estimated, documented and maintained in the study records. The presence and level of mucus was estimated and scored in accordance with a four - step grading system based on the opacity of vaginal lavage specimens.
The level of mucus present in lavage specimens is not summarized within this study report, because of extensive inter - animals variation which did not provide interpretable data. In addition, other measurements (oestrous cycle stages, histopathology etc.) yielded more commonly employed datasets that could be more readily interpreted. An analysis of the female oestrous cycle patterns was reported. The following parameters were analysed and reported for each oestrous lavage sampling interval (i.e. Weeks 3 - 4, 7 - 8, 11 - 12, 16 - 17, 20 - 21, 24 - 25, 29 - 30 - 31, 34 - 35, 38 - 39, 42 - 43, 46 - 47, 50 - 51, 55 - 56 - 57, 60 - 61, 64 - 65, 68 - 69, 73 - 74 and 77 - 78 - 79, corresponding to sampling intervals 1 - 18, respectively).

OESTROUS CYCLE LENGTH
Oestrous cycle length was determined by counting the number of days from the first metestrus [M] or dioestrus [D] in a cycle to the first M or D in a subsequent cycle. Only complete oestrous cycles were used for calculation of oestrous cycle length (i.e., the total number of returns to M or D from oestrus [E] or proestrus [P] from the beginning until the end of the sampling interval). The mean length of the oestrous cycle and the number of complete oestrous cycles in each sampling interval were calculated.

THE NUMBER AND PERCENTAGE OF DAYS IN EACH STAGE OF THE OESTROUS CYCLE
In each sampling interval, the number and percentage of days in E, P, E+P, D, M and D+M was calculated. These data were used to analyse the distribution of cycle states (see below) across the duration of the study.

THE MAXIMUM NUMBER OF CONSECUTIVE DAYS IN AN ESTROGENIC (E OR P) OR ANOESTRUS (D OR M) STATE
For each sampling interval, the number of consecutive days that an animal spends in an estrogenic or anoestrus state was reported. These data were also used to analyse the distribution of cycle states (see below) across the duration of the study.

OESTROUS CYCLE STATES
In each interval, each animal was assigned into 1 of the following categories based on its oestrous cycle status: Regular Cycler: An animal which displays regular 4 - to 5 - day oestrous cycles during the sampling interval. Any animal displaying 2 or more regular cycles in a 14 day sampling interval, or 3 or more regular cycles in a 21 day sampling period was considered a regular cycler. Continuous Oestrus: An animal which remains in an estrogenic state (E or P) for the duration of the sampling interval (14 or 21 days).
Abnormal Cycler – Persistent Oestrus: An animal which displays >3 consecutive days in an estrogenic state (E or P) and which remains in an estrogenic state for >50% of the duration of the sampling interval. Any animal in continuous oestrus, as defined above, was not included in this category.
Continuous Dioestrus: An animal which remains in an anoestrus state (D or M) for the duration of the sampling interval (14 or 21 days).
Abnormal Cycler – Persistent Dioestrus: An animal which displays >5 consecutive days in an anoestrus state (D or M) and which remains in an anoestrus state for >80 % of the duration of the sampling interval. Any animal in continuous dioestrus, as defined above, was not included in this category.
Minor Abnormalities: Any animals which cannot be categorized as a regular or abnormal cycler, or is not in continuous oestrus or dioestrus (i.e. all remaining animals with minor irregularities in oestrous cyclicity).

NON - TERMINAL BLOOD COLLECTION FOR HORMONE ANALYSIS
On Study Day 28 at 0900 hours (± 2 hours), non - terminal blood samples for hormone analysis (prolactin, leptin, and adiponectin) were collected from 20 females/group in the Week 13 Cohort. Blood (approximately 0.8 mL/animal) was collected via the jugular vein into chilled tubes containing sodium heparin. Blood samples were maintained on wet ice, protected from light, until centrifugation. Plasma was isolated in a refrigerated centrifuge, flash frozen in liquid nitrogen, and stored at approximately - 70 °C. The plasma samples were shipped on dry ice via overnight courier for analysis.

Sacrifice and pathology:

POST-MORTEM INVESTIGATIONS
UNSCHEDULED DEATHS
Females that were found dead after 13 Jan 2016 and 1 female removed from study were discarded without examination. Females found dead before 13 Jan 2016, females euthanised in extremis, and 1 female removed from study were euthanised by carbon dioxide inhalation and the following tissues and organs were examined macroscopically, retained in 10 % neutral - buffered formalin, and examined microscopically: fat pads (genital, perineal, and inguinal)*, liver*, ovaries (2), pituitary gland, skin with mammary gland, uterus (with oviducts and cervix), vagina.
* Not examined microscopically

TERMINAL BLOOD COLLECTION FOR HORMONE ANALYSIS (WEEKS 13 AND 26 AND WEEK 52, 66, AND 80 [SUBSET 1])
Each surviving female in the Week 13 and 26 cohorts and surviving Subset 1 females in the Week 52, 66, and 80 Cohorts were euthanised by decapitation (at 0900 hours ± 2 hours) and approximately 3 mL of trunk blood was collected.

MACROSCOPIC EXAMINATION, ORGAN WEIGHTS, AND TISSUE COLLECTION (WEEKS 13 AND 26)
Following decapitation for trunk blood collection for Week 13 and 26 Cohort females, the whole brain (excluding olfactory bulbs) was collected, weighed, rapidly flash frozen by placement in dry ice, and stored at approximately - 70°C for brain microdissection The tissues listed in the table below were examined macroscopically, collected, weighed and immersion fixed in 10% neutral - buffered formalin for histopathological examination, except as noted.
fat pads (genital, perineal, and inguinal)*; liver #; ovaries (2) @; pituitary gland; skin with mammary gland $; uterus (with oviducts and cervix); vagina $
* - Fat pads were dissected and weighed together as 1 unit and then flash frozen in RNAse - free tubes by immersion in liquid nitrogen.
# - After weighing and sampling for histopathology, the remaining liver tissue was processed as follows: A single liver tissue section (approximately 4 mm thick) was taken from the left lateral lobe, divided in 2 and flash frozen in RNAse - free tubes by immersion in liquid nitrogen. The remaining liver tissue was then sectioned into 4 pieces, placed in 4 individual tubes, and flash frozen in liquid nitrogen.
@ - Paired organs were weighed together.
$ - Not weighed.
All frozen tissues were stored at approximately – 70 C for future possible bioanalysis. All formalin fixed tissues remained in formalin for no more than 48 hours and were then embedded in paraffin. The remainder of the carcass was discarded without further examination.

MACROSCOPIC EXAMINATION, ORGAN WEIGHTS, AND TISSUE COLLECTION (WEEKS 52, 66, AND 80 [SUBSET 1])
Following decapitation for trunk blood collection for Subset 1 females in the Week 52, 66, and 80 Cohorts, the whole brain (excluding olfactory bulbs) was collected, weighed, rapidly flash frozen by placement on dry ice, and stored at approximately -70 °C for brain microdissection. The following tissues were examined macroscopically, collected, weighed, flash frozen in RNAse-free tubes by immersion in liquid nitrogen, and stored at approximately -70 C for future possible bioanalysis, except as noted.
Fat pads (genital, perineal, and inguinal); liver *; ovaries (2)#; pituitary gland @; skin with mammary gland $; uterus (with oviducts and cervix); vagina $;
*- A single liver tissue section (approximately 4 mm thick) was taken from the left lateral lobe, divided in 2 and flash frozen in RNAse-free tubes by immersion in liquid nitrogen. The remaining liver tissue was then sectioned into 4 pieces, placed in 4 individual tubes, and flash frozen in liquid nitrogen.
# Paired organs were weighed together.
@- The pituitary gland was examined macroscopically, collected, weighed, and immersion fixed in 10 % neutral-buffered formalin for histopathological examination. Tissue remained in formalin for no more than 48 hours and was then embedded in paraffin.
$- Not weighed.
The remainder of the carcass was discarded without further examination.

MACROSCOPIC EXAMINATION, ORGAN WEIGHTS, AND TISSUE COLLECTION (WEEKS 52, 66, AND 80 [SUBSET 2])
Each surviving Subset 2 female in the Week 52, 66, and 80 Cohorts was deeply anesthetized by intraperitoneal injection of sodium pentobarbital and perfused in situ (at 0900 hours ± 2 hours) with 4.0% paraformaldehyde solution in 0.1 M phosphate buffer via the left ventricle of the heart. The brains were removed (including olfactory bulbs), weighed, and embedded in paraffin for immunohistochemistry, in situ hybridisation, and stereology. The following tissues were examined macroscopically and retained in 10% neutral-buffered formalin for histopathological examination: Fat Pads (genital, perineal and inguinal); Liver; Ovaries (2); Pituitary gland; Skin with mammary gland; Uterus (with oviducts and cervix); Vagina.
Retained tissues remained in fixative for no more than 48 hours and were then embedded in
paraffin. The remainder of the carcass was discarded without further examination.

BRAIN SECTIONING AND MICRODISSECTION
For the isolation, extraction, and analysis of the median eminence (ME) for determination of concentrations of dopamine (DA) and its metabolite, DOPAC (3,4-dihydrophenylacetic acid), frozen brains were trimmed on dry ice or on a cold plate, as needed, and mounted with Tissue-Tek O.C.T. embedding compound, or suitable alternative, on a cryomicrotome sample stage for sectioning. The brain was sectioned in a cryomicrotome set to maintain -10 °C or colder, into a series of coronal brain slice sections (approximately 300 μm thickness) starting anterior to and continuing through posterior to the expected level of the median eminence (ME). The level of the ME was determined visually following proper ventral/dorsal and lateral orientation of the brain based on morphological landmarks (Paxinos and Watson, 2007). The coronal brain slice sections were arranged in a series on chilled microscopic slides (stored in the cryomicrotome) in a manner to demonstrate the progression, anterior to posterior, of the serial coronal sections through the brain, and thereby allowing for identification and dissection of the ME. The sections and slides remained in the cryomicrotome or on dry ice until transferred for dissection and sampling of the ME.
On the same day the brain sectioning was performed, the slides were placed onto a chilled aluminium block (dry ice placed into the well), and the ME was identified under a dissecting microscope. Four sections that best encompassed the ME were selected. The arcuate nucleus was removed from all 4 sections with a chilled 750 μm punch, (leaving the ME behind) and all 4 punches were transferred to a uniquely labelled tube containing 50 μL of RNALater. Tissues were stored refrigerated (2 °C to 8 °C) for at least 18 hours prior to being transferred to a freezer set to maintain temperatures between -65 °C to -75 °C. Using a microdissection tool, the ME from all sections was dissected and placed into a chilled, 0.2-mL centrifuge tube containing 50 μL of HPLC extraction buffer (0.05 M Na2HPO4, 0.03 M Citric acid at pH 3). After collection of the ME, the tubes were placed on dry ice until transferred to a freezer for storage at approximately -70 °C until shipped on dry ice to RTI International, Research Triangle Park, NC, for analysis.
During microtomy at Week 26, Female Nos. 7267 and 7206 in the 500 ppm test substance group did not have slides collected for Level Nos. 19, 20, and 21 of the brain. These brains were damaged at the median eminence during sectioning.

HISTOPATHOLOGY AND MICROSCOPIC EVALUATION
After fixation, protocol-specified tissues were trimmed. Trimmed tissues were processed into paraffin blocks, sectioned according to Charles River SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin. Microscopic examination was performed on the ovaries, pituitary, mammary gland, uterus, cervix, and vagina from all animals euthanised in extremis (including 1 female removed from study) and at the scheduled necropsies. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.
Upon completion of the histopathologic assessment of all tissues a histopathology peer review was performed. Details of the number of animals and tissues examined are described below.
(1) Whole animals (fat pads, liver, ovaries, pituitary gland, skin with mammary gland, uterus [with oviducts and cervix], vagina): All animals in all groups and cohorts (aside from Subset 1 of Weeks 52, 66, and 80, in which only the pituitary gland was saved for histopathological evaluation).
(2) In addition, all report versions, macroscopic abnormalities, organ weights (where applicable), and preliminary immunohistochemistry/stereology data/reporting was reviewed.

IMMUNOHISTOCHEMISTRY, IN SITU HYBRIDISATION, AND STEREOLOGY
Perfused brains from all Subset 2 animals (week 52, 66 and 80 cohorts) were used for immunohistochemistry, in situ hybridisation, and stereology. Perfused brains were shipped in situ (in the skull) by overnight courier on each day of necropsy to PAI Durham. Upon receipt, brains were removed from the skull (no more than 32 hours post-necropsy), weighed, placed into a rat brain trimming matrix, and sectioned once coronally at the area of the median eminence (ME). Both pieces of brain were placed cut-side down into the same block and processed in paraffin on the same day (day of trimming). Upon removal of the brains, the pituitary was also removed from the skull and embedded in paraffin within 48 hours of necropsy. Paraffin blocks were transferred back to the testing facility and retained with the remainder of the protocol-specified paraffin-embedded tissues for histological processing and microscopic examination.
Week 52 Cohort: Paraffin embedded tissues were sectioned (approximately 4 - 5 μm thickness). One section was stained with hematoxylin and eosin and examined by the study pathologist to confirm the correct location in the brain (median eminence). Additional sections were placed on charged slides for immunohistochemistry and SuperFrost Plus slides for in situ hybridisation. Immunohistochemistry and in situ hybridisation (using RNAscope)
Weeks 66 and 80 cohorts: Stereological sectioning of paraffin embedded brain tissue was commenced when the ventral 1/3 of the brain was visible on the face of the paraffin block while in the microtome chuck (tissue prior to achieving this was discarded as it did not contain the arcuate nucleus), and consecutive 5 micrometer thick sections (physical disectors) were obtained and placed onto charged slides. The remainder of the brain containing the arcuate nucleus was exhaustively sectioned, collecting physical dissectors at regular intervals, in order to obtain 8 - 10 sections through the region of interest, ideally located on 5 slides (2 brain sections per slide). The sampling interval was determined in a pilot study using stock animal brains that had been trimmed the same way as the study tissue. At each interval, a single section was taken and stained with hematoxylin and eosin, 3 backup sets of dissectors were obtained and placed onto charged slides, and 2 single sections per interval were taken and placed onto SuperFrost Plus slides for possible future use. Stained slides from each sampling interval were evaluated by the study pathologist. Dissector pairs from all intervals containing the arcuate nucleus were immunohistochemically stained for tyrosine hydroxylase and used for stereological analysis. Slides were scanned at 20x magnification using the Hamamatsu Nanozoomer whole slide scanner and imported into the Visiopharm software. The arcuate nucleus was outlined and Fractionator sampling of a defined percentage of the arcuate nucleus was performed in duplicate, with a goal of counting 100-200 neurons per animal. The number of tyrosine hydroxylase positive neurons within the arcuate nucleus was determined, using the nucleus as the unique counting feature.

Other examinations:

DATA ACQUISITION AND ANALYSIS
ACQUISITION AND REPORTING
The major computer systems used on this study include, but are not limited to, the following systems. All computerised systems used for data collection during the conduct of this study have been validated (with the exception of Microsoft Office); when a particular system has not satisfied all requirements, appropriate administration and procedural controls were implemented to assure the quality and integrity of the data. Knime software was used for calculation and tabulation of oestrous cycle data. These systems were not validated. However, the data generated by these systems were verified to be correct. Therefore, this did not affect the quality or integrity of the study.

Statistics:

STATISTICAL ANALYSIS
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and/or the number of animals or cages (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group.
Body weight, body weight change, food consumption, food efficiency, absolute organ weights (excluding brain weights for Subset 2 animals), morphometry data, and stereology data were subjected to a parametric one-way ANOVA followed by Dunnett's test to compare the test substance-treated groups to the control group, or a two-sample t-test to compare the test substance 2-treated group to the control group. Histopathological findings (non-neoplastic, total incidence combining all severity grades) in test substance treated groups (Group 2 and 3) and Group 4 were compared to the control group (Group 1) by the Fisher’s Exact test. Brain weights collected for animals assigned to Subset 2 were not analysed statistically.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food efficiency

serum/plasma hormone analyses

other: oestrus cycle length, dopamine, body fat

Remarks on result:

other: Mean dietary equivalent to 28 mg/kg bw/day

Dose descriptor:

NOAEL

Remarks:

Carcinogenicity

Effect level:

> 3 000 ppm

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Mean dietary equivalent to 194 mg/kg bw/day

Critical effects observed:

no

Mortality:

No test substance-related observations were noted.

 

Moribundity:

No test substance-related observations were noted.

 

Clinical observations:

No test substance-related observations were noted.

 

Body weight gains:

Test substance-related lower mean body weight gains versus controls were noted for the 3000 ppm test substance group beginning with the first week of test diet administration and continuing throughout the treatment period.

 

Food efficiency:

Test substance-related lower food efficiency versus controls were noted for the 3000 ppm test substance group beginning with the first week of test diet administration and continuing throughout the treatment period.

 

Food consumption:

Food consumption highly variable in the 3000 ppm test substance group (Group 3), with both increases and decreases, but a more consistent trend of decreased food consumption was seen in the last 4 months of the study.

 

Body weights:

Absolute mean body weights for 3000 ppm test substance group (Group 3) were 5 % lower than controls by Study Day 21 and 20.6 % lower than controls at study termination (Study Day 560). These effects were considered test substance-related and adverse. Mean body weight gains in the 500 ppm test substance group and 3000 ppm Group 4 groups were generally comparable to or slightly lower than the control group during the first 12 months of the study, and mean food consumption was generally comparable to the control group throughout the treatment period. Cumulative body weight gains for these groups were numerically or statistically significantly lower than control during the last 6 months of the study. As a result, absolute mean body weights, during the last 6 months of the study, were up to 5.3% and 7.8% lower than the control group, respectively in the 500 ppm test substance group (Group 2) and 3000 ppm Group 4. Due to the late onset and small magnitude of the change these differences were considered test substance-related, but minimally adverse.

 

 

Dose levels:

Target dietary concentrations of Groups 2, 3 and 4corresponded to actual average consumed dose levels of 40, 243 and 236 mg/kg/day, respectively, during the first 91 days of treatment. As the animals reached adulthood and beyond, the overall pattern of lower food efficiency was also reflected in lower actual test substance consumption. Over the entire study, actual average consumed dose levels were 28, 194 and 176 mg/kg/day for Group 2, 3 and 4

dose groups, respectively.

 

Oestrous cycle:

Mean oestrous cycle lengths of approximately 4.0 days, with approximately 2 complete cycles during each 2-week sampling interval were observed for the majority of animals irrespective of treatment group through Week 25. Beginning with Week 29 and continuing through Week 51, the control group showed a tendency for fewer number and percentage of animals with complete oestrous cycles, and a gradual increase in the mean oestrous cycle length. During this time period, there was a tendency for Group 3 animals to have shorter mean cycle lengths and more animals with complete cycles compared to the controls; this pattern was not observed in Groups 2 and 4. Higher mean cycle lengths (between 5 - 8 days) and fewer complete cycles during each sampling interval were noted across all groups from Week 51 through Week 79 of the study.

Complete oestrous cycles were determined by counting the total number of returns to a non-oestrogenic state (M or D) from an oestrogenic state (E or P) from the beginning until the end of the sampling interval).

In the control group, the percentage of days in an estrogenic state (%E + %P) gradually increased between Weeks 7 - 8 and Weeks 42-43, with a peak value of 59.8 % occurring during Weeks 42 - 43. During the same period, the percentage of control females, per sampling interval, with assigned cycle states of persistent or continuous oestrous also showed a steady increase (< 1 % increasing to 50.5 % of females). Subsequently, a slow decline in the percentage of days in an estrogenic state and an increase %D + %M cycle measurements was noted for the remainder of the study, which is considered to reflect a further transition of control female Wistar rats into reproductive senescence that was characterised by a gradual increase in persistent or continuous dioestrus. In contrast, animals in the 500 ppm and 3000 ppm test substance treated animals (Groups 2 and 3) continued to show higher percentages of days in an estrogenic state until later time intervals. Peak values of %E + %P for these groups were achieved at Weeks 50 - 51 (63.5 %) and at Weeks 55 - 57 (67.3 %), respectively, indicating both a longer time in an estrogenic state, and a higher peak value compared to controls. For the Group 4, the peak value for %E + %P was noted at Weeks 46-47 (65.4%), and therefore the differences versus controls for Group 4 were less pronounced than those of the 3000 ppm test substance group.

During the time interval from Weeks 7 - 8 to Weeks 42 - 43, a continuous increase in the percentage of females, per sampling interval, with the assigned cycle states of persistent or continuous oestrus was observed for the groups 2, 3 and 4 (~1 % increasing to 48.08 % and 48.54 % respectively); this transition was the same as the control animals for these two groups. In contrast, the 3000 ppm test substance group (Group 3) displayed a more gradual increase during this time period, and had a statistically significantly lower number of females showing persistent or continuous oestrus during Weeks 34 - 35 (28.8 %) and 38 - 39 (25.7 %). At Weeks 42 - 43 of treatment, 40 % of females in the 3000 ppm test substance group (Group 3) were displaying persistent or continuous oestrus compared to 50. 5% in the control group.

During Weeks 43 - 79, the second half of the treatment period, there was a gradual decline in the percentage of days in an estrogenic state (%E + %P) in the control group, and the percentage of females showing continuous or persistent oestrus decreased from 50.48 % to 12.5 %. In contrast, for females administered 500 ppm or 3000 ppm test substance, the percentage of females showing persistent or continuous oestrus continued to increase through week 57 of treatment, peaking at 61 % of females in each group, followed by a notably slower decline in this percentage in comparison to the controls. For females in Group 4, the highest percentage of females showing persistent or continuous oestrus (59.22 %) was observed during Weeks 46 - 47, which was closer to the peak time interval for the controls (Weeks 42 - 43). After reaching a maximum, the Group 4 animals showed a decline in the percentage of females with persistent or continuous oestrus through week 79 of the study, and the rate of this decline was intermediate between that of the control group and the test substance-treated groups. There was no consistent effect of different treatments on the percentage of regularly cycling animals over the course of the study. An age related decline in the proportion of regularly cycling females was seen across all groups.

 

Fat pad weights:

Test substance-related lower fat pad weights were noted for the 500 and 3000 ppm test substance groups as early as Week 13 and for Group 4 at later time points. The magnitude of change in fat pad weights was greatest for the 3000 ppm test substance group (maximal at Week 52, -50% for organ weight relative to body weight; -59 % for absolute organ weight). These differences persisted until the end of the study and reflected lower amounts of body fat in the treated animals when compared with controls.

 

Leptin and prolactin levels:

Control rats demonstrated an age-related increase in the plasma levels of leptin and prolactin levels, which was also observed for animals in the 500 ppm test substance group, and to a lesser extent in Group 4, but was prevented by the treatment with test substance at 3000 ppm (Group 3). Animals in the high dose test substance group had numerically or statistically significantly lower prolactin values than the control group following 66 and 80 weeks of treatment, and statistically significantly lower leptin values compared to controls following 52, 66 and 80 weeks of treatment. Leptin values were numerically lower for the 3000 ppm test substance group beginning at Day 28, and all of these differences were considered to reflect an effect of treatment.

 

Dopamine and dihydroxyphenylacetic acid levels:

Mean dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) concentrations in the median eminence of the hypothalamus were statistically significantly higher than the control group only at certain time intervals for animals in the 3000 ppm test substance group. For DA, mean values were only statistically significantly higher (+26.3 %) at Week 26. For DOPAC, mean values were statistically significantly higher at Week 52 (+29.4 %) and at Week 80 (+30.0 %), although DOPAC values were numerically higher than control at Week 26 (+31.0 %) and at Week 66 (+15.9 %) in the 3000 ppm test substance group. There were no statistically significant differences in dopamine and DOPAC levels for animals in the 500 ppm test substance and Group 4 at any necropsy interval. Furthermore, there were no clear indications of any consistent differences between the ratio (DOPAC/DA) in comparing the control and treated groups at any necropsy interval; therefore, this measure of dopamine turnover in the median eminence was unaffected by treatment. In the controls and the treated groups, there was a slight decrease in DA and DOPAC levels between Week 13 and Week 26, but the levels of these analytes in the median eminence remained at fairly similar levels from Weeks 26 - 80.

 

Gross macroscopic observations:

There were no test substance-related gross macroscopic observations noted at scheduled necropsy. There were also no definitive adverse test substance-related histologic changes across all time points, and there were no apparent test substance-related effects on proliferative lesions in the uterus, cervix, and vagina. Some histologic differences observed at particular time intervals were suggestive of a difference between groups in the extent of oestrous cycling and/or the progression of animals into reproductive senescence. For example, at Week 26 the 3000 ppm test substance group displayed a statistically significantly lower incidence of “reproductive cycle alteration” (the inability to assign an oestrous cycle stage due to the onset of reproductive senescence) and a lower incidence of age-related atrophy in the ovaries, indicative of continued oestrous cycling in the 3000 ppm test substance group. These differences were not apparent in the 500 ppm test substance group (Group 2) or Group 4. At later time points (Weeks 66 and 80), lower incidences of mucification of the cervix and vagina, and atrophy of the vagina and/or uterus were observed in the 3000 ppm test substance group, and to a lesser extent in the other test substance-treated groups. These differences were also suggestive of differences in oestrous cycling and progression into reproductive senescence between the treated and control groups.

In addition, the incidence of lobuloalveolar hyperplasia of the mammary gland was lower in the 3000 ppm test substance group when compared with the control group at Weeks 52 and 66, with a lesser effect observed in the 500 ppm test substance group (Group 2) and Group 4. However, the trends were not constant across all time points, with similar incidences occurring in control and treated groups at one or more time points.

There was no difference in the amount of tyrosine hydroxylase staining in the arcuate nucleus by immunohistochemistry (for protein) orin situhybridisation (for RNA) between control and test substance-treated groups at Week 52. There were also no test substance-related differences in the number of tyrosine hydroxylase-positive (dopaminergic) neurons in the arcuate nucleus between control and test substance-treated groups as determined by unbiased stereology at Weeks 66 and 80.

 

Table 1 Summary of Results

	Group 1	Group 2	Group 3	Group 4
Clinical Observations
Terminal Body Weights (Study Day 560)		-5.3 %	-20.6 %	-7.8 %
Body Weight gains		↓ Weeks 52 80	↓↓ Weeks 0 - 80	↓ Weeks 52 80
Food Consumption			↓↓ Weeks 60 -80
Food efficiency		↓ Weeks 13 - 80	↓↓ Weeks 0 - 80	↓ Weeks 13 80
Oestrous Cycle length - Weeks 0 - 51 (days)	3.9 - 4.8	4.0 - 4.9	4.0 - 4.7	4.0 - 4.7
Oestrous Cycle length  Weeks 52 - 80 (days)	5.5- 7.7	5.3 - 7.8	4.7 - 6.1	5.1 - 8.3
Animals With Complete Cycles -Weeks 0 - 25 @	86.4 % - 100 %	82.4% - 99.3%	89.6% - 100%	88.8% - 99.3%
Animals With Complete Cycles  Weeks 29 - 57 @	50.5 % - 84.8 %	44.2% - 81.0%	48.6% - 93.3%	46.6% - 78.8%
Animals With Complete Cycles  Weeks 60 - 80 @	37.5 % - 44.8%	26.7% - 62.1%	24.2%- 62.5%	40.9%-  60.6%
Peak Oestrogenic State (%E + % P)	59.8 %	63.5 %	67.3 %	65.4 %
Timing of peak oestrogenic state	Weeks 42- 43	Weeks 50 - 51	Weeks 55 - 57	Weeks 46 -47
Fat Pads Weights		↓	↓↓	↓
Liver Weights		↑	↑	↑
Plasma prolactin age related increase	↑↑	↑		↑
plasma leptin age related increase	↑↑	↑		↑
plasma Adiponectin
Median eminence - Dopamine			↑ Week 26
Median eminence  -DOPAC			↑ Weeks 52 & 80
DO AC/DA Ratio
Macroscopic Observations
Microscopic Observations	See text	See text	See text	See text

@ = Values for Sampling Intervals 7, 13 and 18 adjusted for the longer 3 week collection period (vs. 2 weeks for all remaining sampling intervals)

Conclusions:

The no observed adverse effect level was 500 ppm, equivalent to an average daily intake of 28 mg/kg body weight for females. The test substance did not present carcinogenic properties in rats.

Executive summary:

 Four groups of female Han Wistar (Crl:WI[Han]) rats (145 per group) were administered either basal diet, test substance, or a soil and plant metabolite of the test substance in a GLP compliant study. Test material was given continuously in the diet from Study Day 0 to euthanasia (up to 80 weeks). Target test substance concentrations were 0, 500, 3000 ppm, corresponding to 0 (Group 1), 28 (Group 2), 194 (Group 3) mg/kg body weight/day, respectively. The concentration of the test material metabolite was 3000 pm, corresponding to 194 (Group 4) mg/kg body weight/day. Females were divided into Cohorts and Subsets for euthanasia and post-mortem activities conducted during Study Weeks 13, 26, 52, 66, and 80.

All animals were observed twice daily for mortality and moribundity. Detailed physical observations were recorded weekly. Body weights and food consumption were recorded weekly for the first 26 weeks of the study, then biweekly thereafter. Daily vaginal lavages for determination of oestrous cycles were performed during 2- or 3-week sampling intervals throughout the study and continuing through the day of scheduled euthanasia for animals scheduled for each scheduled necropsy. Blood samples for hormone evaluations (prolactin, leptin, and adiponectin) were collected from Week 13 cohort females on Study Day 28 (non-terminal), from all females at the scheduled necropsy in Weeks 13 and 26, and from Subset 1 females at the scheduled necropsy in Weeks 52, 66, and 80. All Week 13 and Week 26 cohort females and Subset 1 females in the Weeks 52, 66, and 80 cohorts were euthanised by decapitation, and following blood collection, the brain was weighed and flash-frozen for microdissection and determination of dopamine and DOPAC levels in the median eminence of the hypothalamus; selected additional tissues were collected and processed for microscopic examinations or potential future bioanalyses. Subset 2 females in the Weeks 52, 66, and 80 cohorts were euthanised by in situ perfusion of paraformaldehyde; the brains were weighed and used for immunohistochemistry, in situ hybridisation, and stereology; selected additional tissues were processed for microscopic examinations. Selected tissues were weighed from all females in the Week 13 and 26 Cohorts and Subset 1 females in the Week 52, 66, and 80 Cohorts at termination. Lastly, selected tissues were examined microscopically for all females at termination.

No test substance-related mortality, moribundity, or clinical observations were noted during the study. The data generated in this study showed test substance related decreases in absolute body weights (maximum -20.6 % lower than controls) and an even greater decrease in fat pad weights (maximum -59 % lower than controls) in the 3000 ppm test substance treated group, compared to the control. These lower values in the 3000 ppm test substance group were observed starting at the earliest observation points in the study, and they correlated with treatment-related lower values in plasma leptin concentrations at all measurement intervals. In addition, 3000 ppm test substance treatment delayed age related increases in plasma prolactin concentrations which were most notable after 66 and 80 weeks of treatment. Across the time points in this study, the concentration of dopamine (DA) and DOPAC in the median eminence remained fairly constant in the control animals from Weeks 26 through 80, after decreasing slightly after Week 13. The differences from control in DA and DOPAC concentrations for the treated groups were relatively small and were not statistically significant at all time points, but the only statistically significant differences noted were increases in the 3000 ppm test substance group (Week 26 for DA; Weeks 52 and 80 for DOPAC). The vaginal cytology data combined with the histologic examinations were indicative of animals in the 3000 ppm test substance group experiencing a shift in the timing and magnitude of their transition into reproductive senescence compared to controls. The control Wistar rats showed a gradual change from mostly regular 4-day cycles in young animals, to increasing periods of persistent or constant oestrus (i.e. an estrogenic state) up through approximately Weeks 42-43 of the study, and then a gradual transition to mainly persistent or continuous dioestrus (i.e. a non-estrogenic state). The 3000 ppm test substance were slightly delayed in their progression away from regular cycling and into persistent or continuous oestrus, but they achieved a higher estrogenic state (%E + %P value) and remained in this estrogenic state for a longer period in comparison to the controls. These differences were not as apparent in Group 2 or Group 4. For example, the Group 2 and Group 4 showed a similar shift in the time to peak effect, and the duration that the animals remained in a state of predominantly persistent or continuous oestrus, but these differences versus controls were slightly less pronounced for Group 2 and appreciable less pronounced for the Group 4 in comparison to these experienced for the Group 3 animals. In summation, this study provided a detailed characterization of the time course of body weight change and levels of adiposity, oestrous cycle changes, and correlating parameters (i.e. hormone levels, median eminence dopamine and DOPAC levels, organ weights and histopathology changes) that are experienced by female Wistar rats up through 86 weeks of age. Further, it demonstrated that clear differences in many of these time-dependent parameters were affected by treatment with 3000 ppm test substance (Group 3), while fewer changes and lower magnitude differences were observed in rats in Group 2 or Group 4.

In conclusion, the no observed adverse effect level was 3000 ppm, equivalent to an average daily intake of 194 mg/kg body weight for females. The test substance did not present carcinogenic properties in rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this portion of the study was to quantitate dopamine (DA) and the DA metabolite, dihydroxyphenylacetic acid (DOPAC) in brain tissues from the median eminence of samples from the in-life phase of the study conducted at WIL Research, Ashland, OH, using ultra high pressure liquid chromatography (UPLC) coupled with electrochemical detection (ECD).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

632-619-2

EC Number:

632-619-2

Cas Number:

881685-58-1

Molecular formula:

C20 H23 F2 N3 O

IUPAC Name:

632-619-2

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI[Han]

Sex:

female

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

80 weeks

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

Group 2: Test substance 1. Mean dietary equivalent to 28 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

3 000 ppm

Remarks:

Group 3: Test substance 1. Mean dietary equivalent to 194 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

3 000 ppm

Remarks:

Group 4: Test substance metabolite. Mean dietary equivalent to 176 mg/kg bw/day

No. of animals per sex per dose:

145 per dose were used, arranged in the following cohorts:
Week 13 and 26 animals: 20 females per dose
Week 52, 66 and 80 animals - Subset 1: 20 animals
Week 52, 66 and 80 animals - Subset 2: 15 animals

Control animals:

yes, concurrent no treatment

Results and discussion

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

neuropathology

Remarks on result:

other: Mean dietary equivalent to 28 mg/kg bw/day

2

Dose descriptor:

NOAEL

Remarks:

Neurotoxicity

Effect level:

500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

neuropathology

Remarks on result:

other: Mean dietary equivalent to 28 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

DA AND DOPAC IN MEDIAN EMINENCE

In this study, the notes recorded about any potential issues with the collection or processing of individual samples indicated a somewhat higher number of animals with notations per group at the first collection interval (Week 13), and progressively fewer notations per group at later collection intervals (Weeks 26 - 80). This observation could have contributed to somewhat higher variability in results at the Week 13 interval for dopamine and DOPAC determinations.

Only a small number of statistically significant differences from the control group (Group 1) were observed; these differences all were associated with higher values in Group 3 (3000 ppm test substance).

For DA, mean values were only significantly higher (+26.3 %; p<0.05) than the control group values at Week 26 in Group 3 (3000 ppm test substance).

For DOPAC, mean values in Group 3 (3000 ppm test substance) were significantly higher at Week 52 (+29.4 %; p<0.05) and at Week 80 (+30.0 %, p<0.01). In addition, Group 3 mean DOPAC values were numerically higher than control at Week 26 (+31.0 %) and at Week 66 (+15.9 %), but these differences were not statistically significant.

Differences from the control group in DOPAC concentration for Group 4 (3000 ppm test substance metabolite) were in the range of 5.0 % - 28.0 % above the control group during Weeks 26 - 80, but these differences were not statistically significant. In Group 2 (500 ppm test substance), DA and DOPAC means values during Weeks 26 – 80 were both higher and lower than the control value, and none achieved statistical significance.

 

DOPAC/DOPAMINE RATIOS

Dopamine turnover (measured as the DOPAC/DA ratio) was generally unaffected by treatment at each time interval. The DOPAC/DA ratio for Group 2 (500 ppm test substance) at Week 13 was significantly higher (p<0.05) than the control values, but much greater variability in DA and DOPAC values was observed in Week 13 compared to other time points, particularly for the animals in Group 2. Considering the lack of a dose response, and the lack of any similar differences at Weeks 26 - 80, this one difference in DOPAC/DA ratio at Week 13 was considered to reflect the inter-animal variation and not an effect of treatment in Group 2.

DISCUSSION

In each of the statistically significant events, an elevation in the mean DA or DOPAC concentration in Group 3 (3000 ppm) was observed. The DOPAC values for Group 3 during Weeks 26 – 80 were statistically significantly higher than control at Week 52 and Week 80, and the DA value for Group 3 was statistically significantly higher at Week 26. Values for DA and DOPAC in Group 2 (500 ppm ) and Group 4 (3000 ppm) showed smaller differences vs. control, and none were statistically significant.
In general, dopamine turnover in the median eminence was not affected by treatment at anyof the measured sacrifice intervals. There were no clear indications of any consistent differences between the ratio (DOPAC/DA) in comparing the control and treated groups.

 

Table 1 Summary Statistics (mean, SD)

Week	Group	pg DA/μg Protein		pg DOPAC/μg Protein		Turnover (DOPAC/DA Ratio)
		Mean	SD	Mean	SD	Mean	SD
13	Group 1	76.3	38.1	6.6	3.5	0.093	0.024
	Group 2	52.4	40.9	5.1	2.9	0.125*	0.056
	Group 3	65.1	29.6	6.4	3.0	0.100	0.022
	Group 4	75.1	54.4	7.5	4.9	0.108	0.028
26	Group 1	45.3	12.5	4.2	1.7	0.091	0.026
	Group 2	47.3	15.4	4.5	1.8	0.100	0.042
	Group 3	57.2*	12.8	5.5	1.7	0.099	0.036
	Group 4	53.6	15.2	5.4	2.4	0.104	0.054
52	Group 1	44.6	8.2	3.7	1.1	0.083	0.020
	Group 2	46.4	11.4	3.9	1.2	0.086	0.025
	Group 3	47.8	11.6	4.8*	1.4	0.101	0.028
	Group 4	44.8	10.7	4.1	1.2	0.098	0.045
66	Group 1	55.0	17.4	4.4	1.7	0.085	0.042
	Group 2	49.0	11.9	4.5	1.7	0.092	0.029
	Group 3	55.1	13.6	5.1	1.3	0.096	0.031
	Group 4	57.5	19.9	5.3	1.8	0.096	0.027
80	Group 1	43.4	13.1	4.0	1.3	0.095	0.020
	Group 2	40.6	8.1	3.6	0.7	0.090	0.019
	Group 3	50.7	11.0	5.2**	1.4	0.103	0.019
	Group 4	43.7	13.8	4.2	1.5	0.094	0.016

*, **, = p<0.05, p<0.01, vs group 1 controls at same time point (ANOVA + Dunnett’s test)

 

Table 2 ANOVA with Dunnett’s Test (DA pg/μg)

		ANOVA with Dunnett Correction for Multiple Testing
		pg DA/μg Protein
Week	Comparison	Difference Between Means	Simultaneous 95 % Confidence Limits	pairwise p	F value	Overall p
Week 13	Group 2 - Group 1	-23.85	(-56.91, 9.22)	0.21	1.32	0.28
	Group 3 - Group 1	-11.15	(-44.22, 21.91)	0.75
	Group 4 - Group 1	-1.21	(-33.88, 31.45)	1.00
Week 26	Group 2 - Group 1	2.00	(-9.21, 13.22)	0.95	2.91	0.04
	Group 3 - Group 1	11.86	(0.96, 22.76)	0.03
	Group 4 - Group 1	8.31	(-2.45, 19.07)	0.17
Week 52	Group 2 - Group 1	1.76	(-6.37, 9.88)	0.92	0.40	0.75
	Group 3 - Group 1	3.18	(-4.84, 11.20)	0.66
	Group 4 - Group 1	0.19	(-7.83, 8.20)	1.00
Week 66	Group 2 - Group 1	-6.05	(-18.35, 6.26)	0.50	1.01	0.39
	Group 3 - Group 1	0.08	(-12.23, 12.39)	1.00
	Group 4 - Group 1	2.51	(-10.30, 15.32)	0.94
Week 80	Group 2 - Group 1	-2.79	(-11.98, 6.39)	0.81	2.49	0.07
	Group 3 - Group 1	7.28	(-1.77, 16.34)	0.14
	Group 4 - Group 1	0.30	(-8.88, 9.49)	1.00

 

Table 3 ANOVA with Dunnett’s Test (DOPAC pg/μg)

		ANOVA with Dunnett Correction for Multiple Testing
		pg DOPAC/μg Protein
Week	Comparison	Difference Between Means	Simultaneous 95 % Confidence Limits	pairwise p	F value	Overall p
Week 13	Group 2 - Group 1	-1.57	(-4.48, 1.34)	0.43	1.49	0.22
	Group 3 - Group 1	-0.21	(-3.12, 2.70)	1.00
	Group 4 - Group 1	0.91	(-1.97, 3.78)	0.79
Week 26	Group 2 - Group 1	0.34	(-1.20, 1.87)	0.91	2.12	0.11
	Group 3 - Group 1	1.31	(-0.19, 2.80)	0.10
	Group 4 - Group 1	1.19	(-0.28, 2.67)	0.14
Week 52	Group 2 - Group 1	0.19	(-0.75, 1.14)	0.93	2.83	0.04
	Group 3 - Group 1	1.06	(0.13, 2.00)	0.02
	Group 4 - Group 1	0.41	(-0.53, 1.34)	0.60
Week 66	Group 2 - Group 1	0.05	(-1.22, 1.31)	1.00	1.31	0.28
	Group 3 - Group 1	0.62	(-0.64, 1.89)	0.50
	Group 4 - Group 1	0.89	(-0.43, 2.20)	0.26
Week 80	Group 2 - Group 1	-0.39	(-1.38, 0.60)	0.66	5.32	0.0023
	Group 3 - Group 1	1.19	(0.22, 2.17)	0.01
	Group 4 - Group 1	0.25	(-0.74, 1.23)	0.88

 

Table 4 ANOVA with Dunnett’s Test (DOPAC/DA Ratio)

		ANOVA with Dunnett Correction for Multiple Testing
		Ratio (DOPAC/DA)
Week	Comparison	Difference Between Means	Simultaneous 95 % Confidence Limits	pairwise p	F value	Overall p
Week 13	Group 2 - Group 1	0.032	(0.004, 0.060)	0.02	2.81	0.05
	Group 3 - Group 1	0.008	(-0.020, 0.035)	0.85
	Group 4 - Group 1	0.015	(-0.013, 0.042)	0.42
Week 26	Group 2 - Group 1	0.008	(-0.025, 0.041)	0.88	0.29	0.83
	Group 3 - Group 1	0.007	(-0.025, 0.039)	0.91
	Group 4 - Group 1	0.012	(-0.020, 0.043)	0.68
Week 52	Group 2 - Group 1	0.002	(-0.022, 0.026)	0.99	1.59	0.20
	Group 3 - Group 1	0.018	(-0.006, 0.041)	0.17
	Group 4 - Group 1	0.014	(-0.009, 0.038)	0.34
Week 66	Group 2 - Group 1	0.007	(-0.018, 0.033)	0.84	0.44	0.72
	Group 3 - Group 1	0.011	(-0.015, 0.037)	0.60
	Group 4 - Group 1	0.011	(-0.016, 0.037)	0.65
Week 80	Group 2 - Group 1	-0.005	(-0.019, 0.001)	0.80	1.50	0.22
	Group 3 - Group 1	0.008	(-0.006, 0.022)	0.41
	Group 4 - Group 1	-0.001	(-0.015, 0.138)	1.00

 

Applicant's summary and conclusion

Conclusions:

No effects of treatment with testsubstance was observed 500 ppm or below, hence 500 ppm (equivalent to 28 mg/kg/day) was considered to be the no observable effect level (NOEL) in this study.

Executive summary:

This GLP compliant study has been performed to quantitate dopamine (DA) and the DA metabolite, dihydroxyphenylacetic acid (DOPAC) in brain tissues from the median eminence of samples from the in-life phase of rats during 18 months dietary study. Four groups of female Han Wistar (Crl:WI[Han]) rats (145 per group) were administered either plain diet, test substance or a soil and plant metabolite of the test substance. Test material was given continuously in the diet from Study Day 0 to euthanasia (up to 80 weeks). The dose levels of the test substance were 0, 500, 3000 ppm, corresponding to 0 (Group 1), 28 (Group 2), 194 (Group 3) mg/kg body weight/day, respectively. Animals were also given a soil and plant metabolite of the test substance, at 3000 pm, corresponding to 194 (Group 4) mg/kg body weight/day. Females were divided into Cohorts and Subsets for euthanasia and post-mortem activities conducted during Study Weeks 13, 26, 52, 66, and 80. Neurotransmitters were extracted from median eminence (ME) samples and the analysis has been carried out by using ultra high pressure liquid chromatography (UPLC) coupled with electrochemical detection (ECD).

For DA, mean values were only significantly higher (+26.3 %; p<0.05) than the control group values at Week 26 in Group 3 (3000 ppm test substance). For DOPAC, mean values in Group 3 (3000 ppm test substance) were significantly higher at Week 52 (+29.4 %; p<0.05) and at Week 80 (+30.0 %, p<0.01). In addition, Group 3 mean DOPAC values were numerically higher than control at Week 26 (+31.0 %) and at Week 66 (+15.9 %), but these differences were not statistically significant.

In each of the statistically significant events, an elevation in the mean DA or DOPAC concentration in Group 3 (3000 ppm test substance) was observed. The DOPAC values for Group 3 during Weeks 26 - 80 were statistically significantly higher than control at Week 52 and Week 80, and the DA value for Group 3 was statistically significantly higher at Week 26. Values for DA and DOPAC in Group 2 (500 ppm test substance) and Group 4 (3000 ppm test substance metabolite) showed smaller differences vs. control, and none were statistically significant.

In general, dopamine turnover in the median eminence was not affected by treatment at any of the measured sacrifice intervals. There were no clear indications of any consistent differences between the ratio (DOPAC/DA) in comparing the control and treated groups. A concentration of 500 ppm (equivalent to 28 mg/kg/day) can thus be considered as a no observable effect level (NOEL) in this study.