Reaction products of dimethyl phosphonate with 2-alkyl-2-alkylpropanediol and alkanediol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2017 to 23 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

various deviations with no impact on results or integrity of the study (see Appendix 1, attached)

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid: viscous

Details on test material:

- Appearance/physical state: Clear viscous liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION
- Crl:CD(SD) rats were used as the test system for this study. This species and strain of animal is recognised as appropriate for repeat dose toxicity studies. The Sprague Dawley rat was selected because it is a widely used strain for which significant historical control data are available. The number of animals used was the minimum necessary to yield scientifically meaningful results.
- Crl:CD(SD) rats (40 males and 40 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 17 Mar 2017. The animals were approximately 38 days old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed on the following day.
- Each animal was uniquely identified with a subcutaneous microchip (BMDS system) implanted in the dorsoscapular area. All animals were housed for a minimum 13-day acclimation.
- During acclimation, each animal was observed twice daily for mortality and changes in general appearance or behavioUr.
- Data collection during acclimation began on 18 Mar 2017. Individual body weights and cage food weights were recorded and detailed physical examinations were performed periodically during acclimation.

ANIMAL HOUSING
- Upon arrival, all animals were housed 2 to 3 per cage by sex in clean, solid bottom cages containing ground corncob bedding material (Bed O’Cobs; The Andersons, Cob Products Division, Maumee, OH).
- Animals whose cage mate(s) were removed from the study (morbidity or unscheduled death) were not re-paired and remained individually housed for the remainder of the study.
- The cages were cleaned and changed routinely at a frequency consistent with maintaining good animal health.
- The bedding material is periodically analysed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the objectives of this study. The results of these analyses are maintained at Charles River.
- The animals were temporarily separated as necessary to allow for the performance of protocol-specified activities (see Appendix 1 - Study Protocol and Deviations, attached).
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining oral health of animals, and were sanitised weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River.
- Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinical pathology blood collection when food, but not water, was withheld.
- Municipal water supplying the facility was analysed for contaminants according to SOPs.
- The results of the diet and water analyses are maintained at Charles River. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- All animals were housed throughout acclimation and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 73 °F  5 °F (23 °C  3 °C) and 50 %  20 %, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for
automatic collection on an hourly basis.
- Actual mean daily temperature ranged from 71.7 °F to 74.0 °F (22.1 °C to 23.3 °C) and mean daily relative humidity ranged from 38.9 % to 64.9 % during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- On 23 March 2017 (7 days prior to the initiation of dose administration in males), all available rats were weighed and examined in detail for physical abnormalities. These data were collected using WTDMS and reviewed by the Study Director.
- The animals judged suitable for assignment to the study were selected for use in a computerised randomisation procedure based on body weight stratification in a block design. A printout containing the animal numbers and individual group assignments was generated, and the animals were then arranged into treatment groups and housed in social groups according to the printout.
- Individual body weights at randomisation were within±20 % of the mean for each sex.
- Animals not assigned to study were transferred to the Charles River rat colony.
- The control and 700 mg/kg/day groups (Groups 1 and 5, respectively) each consisted of 10 males and 10 females, and the 35, 175, and 350 mg/kg/day groups (Groups 2, 3, and 4, respectively) each consisted of 5 males and 5 females.
- The animals were approximately 7 weeks old at the initiation of dose administration. Individual body weights ranged from 156 g to 198 g for males and from 136 g to 157 g for females at randomization, and from 213 g to 272 g for males and from 159 g to 194 g for females at the initiation of dose administration.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Polyethylene glycol 400 (PEG400; Lot 2EL0005; Re-test date 31 August 2018)

Details on oral exposure:

PREPARATION
- Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18 °C to 24 °C).
- Test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible, disposable, plastic ball-tipped dosing cannula (Instech Solomon, Plymouth Meeting, PA) once daily for up to 28 consecutive days, through the day prior to the primary necropsy.
- The dose volume for all groups was 5 mL/kg.
- Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dosage.
- Adjusted doses became effective the day of collection of the weekly body weights.
- The first day of dosing was Study Day 0; the first week of dosing was Study Week 0.
- Study group assignment is shown in the table below.
- The dosage levels were determined from results of a previous 14-day oral gavage study3 in rats with this test substance in which dosage levels up to 1000 mg/kg/day were well tolerated (only higher mean body weights, cumulative body weight changes, and liver weights were noted in the 1000 mg/kg/day female group). It was anticipated that the high-dosage level will show test substance-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation. The lower dosage levels were selected at interval that were predicted to be narrow enough to reveal any dose-related trends.
- The selected route of administration for this study was oral (gavage) because the oral route is the intended clinical route of exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING AND ANALYSES
- Test substance formulations have been previously shown to be stable and homogenous over the range of concentrations used on this study for up to 10 days at room temperature (18 °C to 24 °C). Therefore, stability of test substance formulations was not assessed on this study.
- Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 7 and 140 mg/mL dosing formulations. These formulations were of sufficient volume for dosing a group of animals for approximately 8 days.
- Samples for resuspension homogeneity determinations were collected from the top and bottom strata of the 7 and 140 mg/mL dosing formulations following room temperature storage for 9 days.
- Samples for concentration analysis were collected from the middle stratum of the first and last dosing formulation (including the control group).
- One duplicate set was analysed and the remaining duplicate set was stored at room temperature and retained as backup samples.
- All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method using charged aerosol detection.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

35 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

175 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

350 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- Ten males and ten females for control and 700 mg/kg bw/day groups.
- Five males and five females for 35, 175 and 500 mg/kg bw/day groups.

Control animals:

yes, concurrent vehicle

Details on study design:

Study design is summarised in the attached flowchart.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

SURVIVAL
- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
- Animals found dead were examined macroscopically as soon as possible to ensure that tissues were not lost due to autolysis.

CLINICAL OBSERVATIONS
- Clinical examinations were performed at the time of dose administration and 1 to 2 hours
following dose administration. During the recovery period, the animals were observed once daily.
- The absence or presence of findings was recorded for individual animals at the scheduled intervals. - Detailed physical examinations were conducted on all animals 1 week prior to randomisation (±2 days), on the day of randomisation, weekly (±2 days) during the study period, and on the days of the scheduled necropsies.
- Daily observations during the recovery period were not necessary on days when detailed physical examinations were conducted.
- 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.
- Any observations noted outside of the above-specified intervals were also recorded.

BODY WEIGHTS
- Individual body weights were recorded 1 week (±2 days) prior to randomization, on the day of randomisation, Study Day 0 (prior to dosing), weekly (±2 days) during the study period, and on the day prior to the scheduled necropsies (non-fasted).
- Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsies.

FOOD CONSUMPTION
- Cage food weights were recorded once weekly (±2 days) beginning following randomisation and throughout the study period. Food consumption was calculated as g/animal/day for the corresponding body weight intervals.
- When food consumption could not be measured for a given interval (selection for necropsy, etc.), the appropriate interval was footnoted as "NA" on the individual tables.

FOB ASSESSMENTS
- FOB assessments were recorded for 5 animals/sex/group during Study Weeks 3 and 5 (end of dosing and recovery periods, respectively).
- The FOB utilised at Charles River is based on previously developed protocols.
- Testing was performed by the same biologists, whenever possible, without knowledge of the animal’s group assignment.
- The FOB was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70±10 dB.
- All animals were observed for the following parameters as listed in the table below.
- Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer et al. The animal was allowed to grip a T-shaped grip bar with its forepaws and was pulled back gently along a platform until its grip was broken. As the backward locomotion continues, the animal’s hindpaws reach a T-shaped rearlimb grip bar, which it is allowed to grasp and then forced to release by continued pulling. Mark-10 series EG digital force gauges (Mark-10 Corporation, Copiague, NY) were used to record the maximum strain required to break forelimb and hindlimb grip. The average of 3 valid measurements was taken as the animal’s score for each grip strength measure.

MOTOR ACTIVITY
- Motor activity was assessed for 5 animals/sex/group during Study Weeks 3 and 5 (end of dosing and recovery periods, respectively).
- Motor activity, recorded after the completion of the FOB, assessment was conducted using a personal computer-controlled system that utilises a series of infrared photobeams surrounding an amber, plastic rectangular cage to quantify each animal’s motor activity.
- Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals.
- The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams.
- The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70±10 dB.
- Each animal was tested separately.
- Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation.
- Data for ambulatory and total motor activity were tabulated.
- Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY
- Blood and urine samples for clinical pathology evaluations (haematology, coagulation, serum chemistry, and urinalysis) were collected from all animals assigned to the scheduled necropsies (Study Days 28 and 42).
- The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection.
- Blood was collected from a jugular vein and from the vena cava at the time of necropsy. Blood was collected into tubes containing potassium K2EDTA (haematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

HAEMATOLOGY AND COAGULATION
- The parameters listed in the table below were examined.

SERUM CHEMISTRY
- The parameters listed in the table below were examined.

URINALYSIS
- The parameters listed in the table below were examined.

Sacrifice and pathology:

ANATOMIC PATHOLOGY – MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all animals. Animals were euthanised by carbon dioxide inhalation followed by exsanguination.
- The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Except where noted, the tissues and organs listed in the table below were collected and placed in 10 % neutral-buffered formalin.

ORGAN WEIGHTS
- Organs listed in the table below were weighed from all animals at the scheduled necropsies (see Appendix 1 – Study Protocol and Deviations).
- Paired organs were weighed together.
- The organ to final body weight and organ to brain weight ratios were calculated.
- When organ weights could not be determined for an animal (due to weighing error, lost or damaged organ, etc.), group mean values were calculated using the available data.
- The organs for which weights could not be determined were designated as “NA” on the individual report tables.

HISTOLOGY AND MICROSCOPIC EXAMINATION
- After fixation, protocol-specified tissues were trimmed according to Charles River SOPs and the protocol.
- Trimmed tissues were processed into paraffin blocks, sectioned according to Charles River SOPs, mounted on glass microscope slides, and stained with haematoxylin and eosin.
- Microscopic examination was performed on the listed tissues from all animals found dead and, at the primary necropsy, for control and 700 mg/kg/day groups.
- Gross lesions were examined from all animals. In addition, the kidney (males) and non-glandular stomach were identified as potential target tissues and were examined from all animals.
- Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate (see Appendix 1 - Study Protocol and Deviations).
- 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.

Statistics:

See below

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

CLINICAL OBSERVATIONS
- Test substance-related yellow material around the mouth was noted in the 700 mg/kg/day group males and females sporadically during the dosing period.
- Clear material around the mouth, as well as yellow material in the urogenital area, was noted in the 700 mg/kg/day group females. These findings were not present during the recovery period in the 700 mg/kg/day group males and females.
- All other clinical observations in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

SURVIVAL
- There were no unscheduled deaths were attributed to administration of test item.
- Female No. 8395 (0 mg/kg/day control group) was euthanised on Study Day 16 with macroscopic and microscopic findings consistent with gavage error and was considered an accidental death.
- Female No. 8418 (35 mg/kg/day group) was found dead on Study Day 14 and had no macroscopic or microscopic findings; the cause of death was undetermined.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

BODY WEIGHTS
- Body weights were unaffected by test substance administration.
- There were a number of statistically significant differences in males and females throughout the dosing and recovery periods. These changes were not considered to be test substance-related and were attributed to biologic variation because they were of a magnitude commonly observed in Sprague Dawley rats, there was no dose-response, the changes did not persist with subsequent test substance administration and/or only occurred at the recovery necropsy.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

FOOD CONSUMPTION
- Food consumption was unaffected by test substance administration.
- There were no statistically significant differences when the control and test substance-treated groups were compared.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

CLINICAL PATHOLOGY
- Haematology and coagulation parameters were unaffected by test substance administration.
- Differences in haematology and coagulation parameters that were statistically significant were not considered test substance-related and were attributed to biologic variation because there were no correlations with other related clinical pathology changes, no dose-response, and/or the change was of a magnitude commonly observed in Sprague Dawley rats under similar study conditions.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

SERUM CHEMISTRY
- Serum chemistry parameters were unaffected by test substance administration.
- Differences in serum chemistry parameters that were statistically significant were not considered test substance-related and were attributed to biologic variation because there were no correlations with other related clinical pathology changes, no dose-response, and/or the change was of a magnitude commonly observed in Sprague Dawley rats under similar study conditions.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

URINALYSIS
- Test substance-related, statistically significant lower mean pH was noted in the 350 and 700 mg/kg/day group females at the primary necropsy. The difference was not present in the 700 mg/kg/day group females at the recovery necropsy.
- Other differences in urine parameters that were statistically significant were not considered test substance-related and were attributed to biologic variation because there were no correlations with other related clinical pathology changes, no dose-response, and/or the change was of a magnitude commonly observed in Sprague Dawley rats under similar study conditions.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

FOB ASSESSMENTS
- Home cage observations: Home cage observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the Study Week 3 and 5 evaluations.
- Handling observations: Handling observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the Study Week 3 and 5 evaluations.
- Open field observations: Open field observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the Study Week 3 and 5 evaluations.
- Sensory observations: Sensory observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the Study Week 3 and 5 evaluations.
- Neuromuscular observations: Neuromuscular observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the Study Week 3 and 5 evaluations.
- Physiological observations: Physiological observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the Study Week 3 and 5 evaluations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

ORGAN WEIGHTS
- There were no test substance-related alterations in organ weights at the scheduled necropsies. However, some statistically significant differences were observed when the control and test substance treated groups were compared. Lower mean absolute brain weights were noted in the 35 and 700 mg/kg/day group males at the primary necropsy. A dose-response relationship was lacking and there were no microscopic correlates; the differences were attributed to biological variability.
- At the recovery necropsy, higher mean adrenal gland (relative to brain weight) and lower thymus (absolute and relative to final body and brain weights) weights were noted in the 700 mg/kg/day group males and higher mean ovaries/oviducts (absolute and relative to brain weight) weights were noted in the 700 mg/kg/day group females. The differences were not observed at the primary necropsy and weights were within the historical control database range; the differences were attributed to biological variability and not related to administration of test item.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MACROSCOPIC EXAMINATION
- Test substance-related findings in the non-glandular stomach were noted in the 175, 350 and 700 mg/kg/day group males and females at the primary necropsy.
- Findings consisted of thickened stomach and yellow contents, areas and/or discoloration. Thickened stomach correlated microscopically with mucosal hyperplasia and yellow areas/discoloration correlated with hyperkeratosis; yellow contents lacked microscopic correlate. Yellow contents were noted in a single 700 mg/kg/day group female at the recovery necropsy, with no microscopic correlate.

Neuropathological findings:

no effects observed

Description (incidence and severity):

MOTOR ACTIVITY
- Motor activity patterns (total and ambulatory activity counts) were unaffected by test substance administration.
- There were no statistically significant changes for the test substance-treated males and females when compared to the control group at the Study Week 3 and 5 evaluations. Values obtained from the 6 epochs evaluated (0-10 minutes, 11-20 minutes, 21-30 minutes, 31-40 minutes, 41-50 minutes, and 51-60 minutes) and the overall 60-minute test session were comparable to the concurrent control values.
- No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated on Study Weeks 3 and 5.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MICROSCOPIC EXAMINATION
- Incidence of selected histopathologic findings at the Study Day 28 primary necropsy is shown in the attached table.
- Test substance-related microscopic findings were noted in the non-glandular stomach of the 175, 350 and 700 mg/kg/day groups at the primary necropsy, consisting of mucosal hyperplasia and hyperkeratosis.
- Chronic-active inflammation primarily localiSed in the submucosa was additionally noted in the 350 and 700 mg/kg/day group males and in a single 350 mg/kg/day group female. Increased incidence of hyaline droplet accumulation was noted in kidneys of the 175, 350 and 700 mg/kg/day group males.
- Mucosal hyperplasia of the nonglandular stomach was characterised by diffusely thickened stratified squamous epithelium, which correlated with thickened stomach noted macroscopically. Hyperkeratosis was characterised by thickened keratin layer, which correlated macroscopically with yellow discoloration/areas. Within the kidney, hyaline droplet accumulation was characterised by increased incidence of variably-sized brightly eosinophilic intracytoplasmic droplets within proximal convoluted tubular epithelial cells; no correlates were noted.
- Incidence of selected histopathologic findings at the Study Day 42 necropsy is shown in the attached table.
- At recovery, mucosal hyperplasia and hyperkeratosis were noted in the 700 mg/kg/day group males and females with lower incidence and severity grade when compared with the primary necropsy, consistent with partial recovery. Hyaline droplet accumulation was noted in a single control group male, but was absent in the 700 mg/kg/day group males, consistent with recovery.
- There were no other test substance-related histologic changes noted at the scheduled necropsies. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects reported at the highest dose level of 700 mg/kg bw/day

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

ANALYSES OF DOSING FORMULATIONS

- Summary tables showing results of homogeneity and concentration analyses are attached.

- The analysed dosing formulations contained 98.7 % to 109 % of the test substance which was within the protocol-specified range of target concentrations for suspensions (85 % to 115 %) and were homogeneous.

- The test substance was not detected in the analysed vehicle formulation that was administered to the control group (Group 1).

CORRELATIONS OF SELECTED OBSERVATIONS

Necropsy	Organ weight	Clinical pathology	Histopathology
Nonglandular stomach - discoloration/area(s), yellow	Not applicable	Not applicable	Non-glandular stomach - hyperkeratosis
Nonglandular stomach - thickened	Not applicable	Not applicable	Non-glandular stomach - mucosal hyperplasia

Applicant's summary and conclusion

Conclusions:

Oral administration of test item to Crl:CD(SD) rats at dosage levels of 35, 175, 350, and 700 mg/kg/day for up to 28 days was well tolerated at all dosages. All findings were considered to be non-adverse. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 700 mg/kg/day in both males and females.

Executive summary:

GUIDELINE

The study was conducted in accordance with the OECD Guideline for the Testing of Chemicals, Guideline 407, Repeated Dose 28-Day Oral Toxicity Study in Rodents, October 2008. The objectives of the study were to evaluate the potential toxicity of the test item when administered daily by oral gavage to Sprague Dawley rats for 28 consecutive days, and to evaluate the potential reversibility of any findings over a 14-day non-dosing observation period. This study also included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment.

 

METHODS

Test item in the vehicle polyethylene glycol 400 (PEG400) was administered orally by gavage once daily for up to 28 consecutive days to 4 groups (Groups 2, 3, 4, and 5) of Crl:CD(SD) rats. Dosage levels were 35, 175, 350, and 700 mg/kg/day for Groups 2, 3, 4, and 5, respectively. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Groups 1 and 5 each consisted of 10 animals/sex and Groups 2 to 4 each consisted of 5 animals/sex. Following up to 28 days of dose administration, up to 5 animals/sex group were euthanised; the remaining 5 animals/sex in the control and high-dose groups were euthanised following a 14-day non-dosing (recovery) period. For toxicology assessment, all animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body weights and cage food weights were recorded weekly (± 2 days). Functional observational battery (FOB) and motor activity data were recorded for 5 animals/sex/group during Study Weeks 3 and 5 (end of dosing and recovery periods, respectively). Clinical pathology parameters (haematology, coagulation, serum chemistry, and urinalysis) were analysed for all animals assigned to the primary (Study Day 28) and recovery (Study Day 42) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals.

 

RESULTS

There were no test substance-related effects on survival, body weights, food consumption, FOB, motor activity, haematology, coagulation, serum chemistry, or organ weights. There were 2 unscheduled deaths: 1 control group female was euthanised on Study Day 16 with macroscopic and microscopic findings consistent with gavage error, and one 35 mg/kg/day group female was found dead on Study Day 14 with no macroscopic or microscopic findings. Test substance-related yellow material around the mouth was noted in the 700 mg/kg/day group males and females. Clear material around the mouth, as well as yellow material in the urogenital area, was noted in the 700 mg/kg/day group females. There were no other test substance-related clinical observations. Test substance-related lower urine pH was noted in the 350 and 700 mg/kg/day group females at the primary necropsy. The difference was not present in the 700 mg/kg/day group females at the recovery necropsy. Test substance-related mucosal hyperplasia and hyperkeratosis of the non- glandular stomach were noted in the 175, 350, and 700 mg/kg/day group males and females with a higher incidence of minimal hyaline droplet accumulation in the kidneys of the 175, 350, and 700 mg/kg/day group males at the primary necropsy. Mucosal hyperplasia correlated macroscopically with thickened stomach and hyperkeratosis correlated with yellow discoloration/areas; hyaline droplet accumulation had no correlates. Chronic-active inflammation primarily localised in the submucosa was additionally noted in the 350 and 700 mg/kg/day group males and in a single 350 mg/kg/day group female. Yellow contents were noted in a single 700 mg/kg/day group female at the recovery necropsy; however, there were no microscopic correlates. Gastric forestomach findings were partially resolved at the recovery necropsy, and hyaline droplet accumulation was absent at the recovery necropsy, consistent with recovery. Gastric and renal findings were considered to be non-adverse.

CONCLUSION

Oral administration of test item to Crl:CD(SD) rats at dosage levels of 35, 175, 350, and 700 mg/kg/day for up to 28 days was well tolerated at all dosages. All findings were considered to be non-adverse. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 700 mg/kg/day in both males and females.