Fatty acids, tallow, compds. with triethanolamine

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:

14 January 2016 to 23 June 2016

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Official notice of J MHLW, METI and ME (31 March 2011), YAKUSHOKUHATSU No. 0331. 7, SEIKYOKU No. 5, KANPOKIHATSU No. 110331009.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Fatty acids, tallow, compds. with triethanolamine

EC Number:

263-158-1

EC Name:

Fatty acids, tallow, compds. with triethanolamine

Cas Number:

61790-67-8

IUPAC Name:

Fatty acids, tallow, esters of triethanolamine

Test material form:

liquid

Details on test material:

Appearance: Brown liquid
Storage: At ambient temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST.

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The RccHan ™;WIST strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 41 to 47 days.
- Weight at study initiation: 127 to 162 g for males and 115 to 147 g for females. On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ±20 % of the mean for the appropriate sex; no replacements were required.
- Fasting period before study: Not specified
- Housing: Five animals of the same sex (main study and recovery phases) were housed in cages of polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were change weekly, following a rotation plan, to further minimize possible effects of spatial variations.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 12 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. Certificates of analysis were also received from the suppliers of the wood based bedding and Aspen chew blocks. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 ºC
- Humidity (%): 40-70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated. There was a minimum of 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Administration was by oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth. Animals were treated at constant doses at a volume of 5 mL/kg bw, calculated from the most recently recorded scheduled body weight.
The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test material was weighed into a suitable container. Vehicle was added to the mixture (to achieve approximately 75 % of the final volume) and magnetically stirred. The remaining vehicle was then added to make up to the required volume and mixed until homogenous. A series of suspensions at required concentrations were prepared by dilution of individual weightings of the test material. Preparations were made weekly and refrigerated (nominally 2-8 °C).
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

VEHICLE
- Justification for use and choice of vehicle: Not specified
- Concentration in vehicle: At nominal concentrations of 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the vehicle.
Adequate homogeneity and stability of the test item in the vehicle at these concentrations was achieved following refrigerated (2-8 °C) storage for up to 15 days and at ambient temperature (nominally 21 °C) for two days.
Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analyzed for achieved concentration of the test material.

PREPARATION OF CALIBRATION STANDARDS
A primary standard solution (1000 µg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of test material in Ethanol (50 mL). Solutions for instrument calibration were prepared by appropriate dilution of the primary standard using Ethanol and contained test material at nominal concentrations of 100 ng/mL, 200 ng/mL, 400 ng/mL, 600 ng/mL, 800 ng/mL and 1000 ng/mL. Calibration standards were matrix-matched by including 200 µL of the initial vehicle extract.
Calibration solutions were analyzed by liquid chromagraphy with mass spectrometric detection (LC-MS/MS) at the beginning of each sample analysis sequence as a minimum using the conditions detailed in the chromatographic section. Sample results were quantified by using a single bracketing standard at the appropriate concentration.

PREPARATION OF TEST SAMPLES
A representative sample of test formulation (1 mL, accurately weighed) was dispersed using ultrasonic vibration in a suitable volume of Ethanol. The extract was further diluted using Ethanol, to provide a solution containing test material at an expected concentration within the range 400 ng/mL to 800 ng/mL.
The concentration of the test material in the final solution was quantified by LC-MS/MS detection as detailed in the chromatographic section.

PREPARATION OF RECOVERY SAMPLES
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (corn oil) with known amounts of test material. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

INSTRUMENTATION PARAMETERS
LC-MS/MS: Agilent 1100 Binary pump, Perkin Elmer PE200. Autosampler and Sciex API 3000 mass spectrometer
Column: Agilent Poroshell SB C18, 2.7 µm, 100 x 4.6 mm
Column temperature: 50 °C
Sample temperature: Ambient
Mobile Phase A: Ethanol/water/formic acid 50/50/0.1 v/v
Mobile Phase B: 0.1 % formic acid in ethanol

Linear Gradient
Time (minute) %A %B
0.0 50 50
0.5 50 50
3.0 0 100
7.0 0 100
7.1 50 50
10.0 50 50

Flow rate: 0.5 mL/minute
Injection volume: 10 µL
Run time: 10 minutes
Needle wash: Propan-2-ol
Mass spectrometer: Sciex API 3000 or equivalent
Ionization: Turboionspray – positive ion mode
Source temperature: +500° C
Collision gas: Nitrogen
Collision energy: 40 eV
Dwell time: 300 ms
Pause time: 5 ms
Ions to be monitored:
m/z 653.0 ¿ m/z 283.4 (used for quantitate)
m/z 653.0 ¿ m/z 309.6 (monitor only)
m/z 679.1 ¿ m/z 309.6 (monitor only)

CALCULATIONS
The peak area response for the test material in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for test material in sample and procedural recovery chromatograms was measured.

The response factor for the single bracketing standard was determined using the following equation:

Response factor (RF) = Calibration standard peak response (Ac) / Concentration of calibration standard (ng/mL)

The concentration of test material was determined using the following equation:

Analysed concentration (mg/mL) = (As/RF) x (V/W) X (D/100 000)

Procedural recovery values were determined using the following equation:

Procedural recovery (%) = (Analysed concentration (mg/mL)/ Fortified concentration (mg/mL)) x 100

Sample concentrations were corrected for procedural recoveries using the following equation:

Corrected concentration, mg/mL = Analysed concentration, mg/mL x (100/R)

Where
Ac = Peak response for test material in single standard.
As = Peak response for test material in sample chromatogram.
RF = Mean response factor of appropriate set of bracketing standard
V = Dilution volume (mL)
W = Sample weight (g)
R = Appropriate procedural recovery value
D = Density of sample (g/mL)

VALIDATION OF THE ANALYTICAL PROCEDURE
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The linearity of detector response over the calibration standard concentration range.
The repeatability of the lowest and highest concentration calibration standards.
The method accuracy and precision, by determining five procedural recoveries at nominal concentrations of 2 mg/mL and 200 mg/mL during the method validation.


HOMOGENEITY AND STABILITY IN CORN OIL FORMULATIONS
The homogeneity and stability of the test material in corn oil formulations was assessed at nominal concentrations of 2 mg/mL and 200 mg/mL, during ambient temperature and refrigerated storage. Freshly prepared specimen formulations (300 mL) were equally sub-divided (3 × 100 mL) into 3 amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

AMBIENT TEMPERATURE STORAGE (nominally +21 ºC)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for 20 minutes (representing 0 hour) and 4 hours, single samples (nominally 1 mL) were removed for analysis from approximately one quarter, one half and three quarters the depth (representing the top, middle and bottom) of the continuously stirred formulation.
The remainder of the bottle was stored at ambient temperature and after 2 and 15 days storage the contents were remixed and sampled as detailed above.

REFRIGERATED STORAGE (nominally +4 ºC)
The remaining bottles were refrigerated on receipt and on Days 2 and 15 the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for a minimum of 20 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.


CONCENTRATION OF DOSE FORMULATIONS
For Week 1 and Week 4, freshly prepared test formulations were sampled (4 × 1 mL, accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate samples were analyzed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.

RESULTS
METHOD VALIDATION
The analytical procedure was successfully validated for the test material in Corn oil with respect to the specificity of chromatographic analysis, the linearity of detector response, repeatability, method accuracy and precision.
The specificity of the HPLC assay was demonstrated by the absence of a peak at the characteristic retention time for the test material in the control sample chromatogram.
Linearity was confirmed over the nominal concentration range 100 ng/mL to 1000 ng/mL (Table 1A) with a correlation coefficient of 0.994;
The repeatability was <4 % for six replicate injections of standard solutions containing the test material at nominal concentrations of 100 ng/mL and 1000 ng/mL;
Method accuracy and precision were confirmed; a mean procedural recovery value of 102 % (CV=2.86 %, n=5) was obtained for 2 mg/mL and 102.3 % (CV=1.55%, n=5) was obtained for 200 mg/mL.

HOMOGENEITY AND STABILITY OF DOSE FORMULATIONS
The homogeneity and stability of the test material in Corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 2 mg/mL and 200 mg/mL.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 4 hours, and on re-suspension following storage at ambient temperature for 2 days at 2 mg/mL and 15 days at 200 mg/mL and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 6 % of the initial time zero value and the coefficient of variation was less than 5 %. The exception was the 200 mg/mL samples after 2 days ambient storage which were +11.8 % from time zero with an 18 % CV.
Recovery results during the trial remained within ±7.5 % of the mean recovery found during validation showing the continued accuracy of the method.


CONCENTRATION OF DOSE FORMULATIONS
The mean concentrations of the test material in test formulations analyzed during the study. The mean concentrations were within applied limits +10/-15 %, confirming the accuracy of formulation.

CONCLUSION
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity and stability was confirmed for the test material in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days (2 mg/mL) and 15 days (200 mg/mL) and refrigerated storage for up to 15 days.
The mean concentrations of the test material in test formulations analyzed for the study were within ±5 % of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Four weeks followed by a two-week recovery period. Cessation of treatment for the recovery phase animals started on the day of necropsy of animals allocated to the main study.

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per dose for the main study.
5 animals per sex for the high dose and control group in the recovery phase.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study were selected in conjunction with the Sponsor.
In an acute oral toxicity study a single oral dose of the test material at a dose level of 2000 mg/kg was well tolerated and did not result in any mortality or signs of systemic toxicity.
In a 14-day repeat-dose range finding study conducted doses of 100, 300 and 1000 mg/kg/day were well tolerated and did not result in any mortality of signs of systemic toxicity.
Based on this information, it was considered appropriate to investigate a high dose level of 1000 mg/kg/day (the limit dose for this study type). All dose levels were expected to be tolerated over a 4-week administration period. The intermediate and low dose levels were selected to assess any dose-responsiveness of any test substance-related finding following a longer (i.e. 4 weeks) treatment period.
- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
- Post-exposure recovery period: 2 week recovery period (15 days).

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
Signs are considered in two parts: Detailed observations recorded at times in relation to dose administration, classified as ‘signs associated with dosing’, and extended changes in condition, classified as ‘clinical signs’.
Clinical observations are presented for each animal that showed signs, providing detail of type of sign, week of occurrence and information on the duration of the sign applicable.

- Clinical and Behavioral Observations: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization and recovery periods, observations of the animals in their cages were recorded at least once per day.

- Signs Associated with Dosing: Observed daily during the treatment period, detailed observations were recorded at the following times in relation to dose administration. During Week 1 of treatment there was a pre-dose observation as well as observations at the end of dosing each group, one to two hours after completion of dosing of all groups and as late as possible in the working day. Observations were also carried out daily during Week 2 of treatment to termination. There was a pre-dose observation as well as observation one to two hours after completion of dosing of all groups.

- Detailed Physical Examination and Arena Observations: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.


BODY WEIGHT: Yes
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy. The last scheduled body weight was recorded on Day 28 for the main study animals (prior to overnight deprivation of food and water for clinical pathology investigations) and Day 14 of recovery for the recovery phase animals.
Group mean weight changes were calculated from the weight changes of individual animals.


FOOD CONSUMPTION: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study. Food remaining for the last week of the treatment and recovery periods was recorded on Day 28 (i.e. prior to overnight deprivation of food for clinical pathology investigations) and Day 14, respectively.
Weekly group mean food consumptions and standard deviations were derived from unrounded cage values. Overall mean food consumption values were calculated from the group mean values.


FOOD EFFICIENCY: No


WATER CONSUMPTION: Yes
Fluid intake was assessed by daily visual observation. No test item-related effects were observed and consequently quantitative measurements were not performed.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
Blood samples were collected on day 29 from all main study animals and on day 15 of recovery from all the recovery animals.
Samples taken from the main study animals on Day 29 were collected after overnight withdrawal of food and prior to dosing. Sampling was performed on the day after overnight collection of urine so the animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Animals sampled on Day 15 of recovery had access to food and water prior to sampling.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC)‡, Differential leucocyte count:‡ (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells) and Platelet count (Plt).
‡ = Recovery phase animals were only examined for these parameters

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples (nominally 0.5 mL) were taken from Main study animals (on Day 29) into tubes containing citrate anticoagulant and examined using an ACL series analyzer and appropriate reagent in respect of:

Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.


CLINICAL CHEMISTRY: Yes
Blood samples were collected on day 29 from all main study animals.
Blood sampling was performed on the day after overnight collection of urine. Animals, were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (gGT), Total bilirubin (Bili), Total bile acids (BiAc), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb).
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.
Albumin to globulin ratio (A/G Ratio) was calculated as:

A/G Ratio = Albumin concentration / (Total protein - Albumin concentration)


URINALYSIS: Yes
Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight on day 29 from all main study animals.
The individual samples were examined for the following characteristics:
- Using manual methods: Clarity and Colour - by visual assessment, Volume (Vol) - using a measuring cylinder, pH - using a pH meter and Specific gravity (SG) - by direct refractometry using a SG meter.
- Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones (Keto), Bile pigments (Bili) and Blood pigments (UBld).
- Using a Roche P Modular Analyzer: Protein (T-Prot), Creatinine (T-Creat), Glucose (T-Gluc), Sodium (T-Na), Potassium (T-K) and Chloride (T-Cl).
A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described: Epithelial cells (Epi), Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A).
The slide was also examined for abnormalities in spermatozoa and crystals.
Group means and standard deviations were presented for volume, pH, specific gravity, protein, creatinine, glucose and electrolytes only.

NEUROBEHAVIOURAL EXAMINATION: Yes
-Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all Main study animals in Groups 2 and 3 and all Recovery phase animals during Week 4 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing. The following measurements, reflexes and responses were recorded:

- Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

- Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

- Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

- Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

- Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed. At any point during the observations, additional comments were made as free text where considered appropriate.

-Motor activity
During Week 4 of treatment (before dosing), the motor activity of all Main study animals in Groups 2 and 3 and all Recovery phase animals was measured using a Rodent Activity Monitoring System.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

IMMUNOLOGY: No

Sacrifice and pathology:

METHOD OF KILL
Carbon dioxide asphyxiation with subsequent exsanguination.

NECROPSY
All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass. The organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 1.


ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals.
Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.

FIXATION
Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of testes which were fixated in modified Davidson’s fluid and eyes which were fixated in Davidson’s fluid.


HISTOLOGY
- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
- Full list: Main study animals of Groups 1 and 4.
- Abnormalities only: All main study animals of Groups 2 and 3 and all recovery phase animals.
- Routine staining: Sections were stained with hematoxylin and eosin.


LIGHT MICROSCOPY
Tissues preserved for examination were examined as follows:
- All tissues specified in table 1 for all main study animals of Groups 1 and 4 at terminal sacrifice
- All tissues with abnormalities from the main study animals of Groups 2 and 3 at terminal sacrifice.
- All tissues with abnormalities from the recovery phase animals at recovery sacrifice.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Statistics:

See below

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The general appearance and behavior of the animals was unaffected by treatment.

Mortality:

no mortality observed

Description (incidence):

There were no deaths throughout the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

A visual assessment of water intake did not reveal any effects of treatment.
Males receiving 100 or 300 mg/kg/day appeared to consume more water than controls on Day 26 but this was considered an incidental finding given its isolated nature and absence of any similar effect at the high dose level in males or at any dose level in females.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test material-related findings.
Slight inter-group differences were evident for some white blood cell parameters at the end of the treatment or recovery periods but all were considered unrelated to administration of the test material. Such differences included a statistically significant increase in mean monocyte count at the end of the treatment period in females given 1000 mg/kg/day. However, based on lack of dose-response and of any correlating finding, and given that no similar trend was evident in males, this was considered not to be treatment-related. Statistically significant decreases were observed in mean lymphocyte, large unstained cells and total white blood cell counts at the end of the recovery period in females previously given 1000 mg/kg/day. However, no similar changes were identified in females at the end of the treatment period and all individual values for control and previously treated females were within historical control ranges (5 to 90-percentile ranges of 2.68-8.53 x10^9/L for lymphocytes, 0.01-0.08 x10^9/L for large unstained cells and 3.47-11.00 x10^9/L for total white blood cell counts; n=140 in all cases) at the end of the recovery period. Consequently, these findings were considered not to be treatment-related.
The slightly lower mean reticulocyte counts seen at the end of the treatment period in males given 300 or 1000 mg/kg/day lacked dose-response and was considered an incidental finding in the absence of any other associated test item-related effects amongst red blood cell parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related findings.
All inter-group differences from controls, including those which attained statistical significance, were generally minor, confined to one sex or lacked dose-relationship. In particular, the marginally low mean triglyceride concentrations seen in all treated groups of males was considered an incidental finding exacerbated by the slightly high values in two control males. Mean sodium concentration was marginally high in males which received 300 or 1000 mg/kg/day but this was due to the variability of results amongst the control and treated groups (two controls, had slightly low values whereas a couple of treated animals, one dosed at 300 mg/kg/day and one dosed at 1000 mg/kg/day, had slightly high values).

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material related findings.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

SENSORY REACTIVITY AND GRIP STRENGTH
Sensory activity and grip strength were unaffected by treatment.

MOTOR ACTIVITY
Motor activity scores were not affected by treatment. There were no statistically significant differences from controls between the total high and low beam scores (rearing and cage floor activity, respectively) achieved over the entire one hour recording period for treated animals. Statistical significance was attained for three of the 6-minute interval scores (1/20 in males and 2/20 in females) but there was no consistency between the sexes and the inter-group differences between the control and high dose animals were therefore attributed to natural variation rather than to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of organ weights revealed slightly high mean absolute and adjusted spleen weight at the end of the treatment and recovery periods in females which received 1000 mg/kg/day. At the end of the treatment period, three high dose females had absolute values that were just above recent historical control ranges (0.318-0.471 g for absolute values; n=25) for the equivalent termination period. At the end of the recovery period, all individual absolute spleen weights for females which had previously received 1000 mg/kg/day were within recent historical control ranges (0.313-0.584 g for absolute values; n=25) and considered comparable to the concurrent control range (0.376 to 0.517 g) for the equivalent termination period.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed after 4 weeks of treatment or 2 weeks of recovery revealed no test material related lesions.
The incidence and distribution of all findings were consistent with the common background seen in Han Wistar rats at the test site.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No changes related to treatment with 1000 mg/kg/day of test material were seen in any tissues.

Histopathological findings: neoplastic:

not examined

Details on results:

The oral administration of test material to Han Wistar (RccHan™;WIST) rats for 4 weeks at dosages up to 1000 mg/kg/day was well tolerated with no adverse effect of treatment. A slight increase in spleen weight in females which received 1000 mg/kg/day was recorded but the finding was not associated with any clear test item-related haematological or histopathological change at the end of the treatment period. Slightly increased extramedullary hemopoiesis was seen in one high dose main study female at microscopic examination, which may have contributed to the reported higher mean weight in this tissue, but this was considered not to be a test item-related change given its isolated occurrence. Spleen weights remained slightly high at the end of the 2-week recovery period in females which had previously received 1000 mg/kg/day, but the difference from control was less than that evident at the end of the treatment period and all individual values were within recent historical control ranges or close to the concurrent control range reported for the recovery females. As no test item-related histopathological change was identified in the main study animals, histopathological examination of this tissue was not required for the recovery phase animals. Overall, this was considered a non-adverse finding given the slight degree of change observed, the absence of any clear histopathological correlate or supportive haematological changes and taking into consideration that partial recovery was evident following a 2-week off-dose period.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test material-related effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg/day.

Executive summary:

The potential for the test material to cause repeated dose toxicity was investigated in a GLP study conducted in accordance to the standardised guidelines OECD 407, EU method B7, OPPTS 870.3050 and Japanese guidelines YAKUSHOKUHATSU No. 0331. 7, SEIKYOKU No. 5 and KANPOKIHATSU No.110331009.

The systemic toxic potential of the test material was investigated in a 4-week oral gavage study in the Han Wistar (RccHan™;WIST) rat. Recovery from any effects was evaluated during a 2-week recovery period. Main study animals received the vehicle (corn oil) or the test material (100, 300 or 1000 mg/kg/day by oral gavage for four weeks. Recovery animals were similarly treated for four weeks followed by a two-week off dose period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

There were no deaths throughout the study period. The general appearance and behaviour, sensory activity, grip strength and motor activity of the animals were not affected by treatment. There was no effect of treatment on body weight gain or food consumption and a visual assessment of water intake did not reveal any suspicion of any treatment related effect. The haematological investigation at the end of the treatment period did not reveal any clear test material-related change. Total and differential leukocyte counts performed on recovery animals also showed no changes related to the previous treatment. The biochemical examination and urinalysis performed at the end of the treatment period did not reveal any test material-related findings. There were no adverse treatment-related effects on organ weights. There were no treatment-related macroscopic or microscopic findings.

It is concluded that oral administration of test material to Han Wistar rats for 4 weeks at dosages up to 1000 mg/kg/day was well tolerated. There were no clear target organs identified at histopathological examination. Consequently, the No Observed Adverse-Effect-Level (NOAEL) was considered to be 1000 mg/kg/day.