Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 08 June 2015 and 04 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it was conducted according to OECD Test Guideline 422 using a screening method and in compliance with GLP.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Strain/Species Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd.
Number of animals ordered: 44 males and 44 females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatisation: Five days before commencement of treatment.
Age of animals at the start of the study
Males: Approximately 70 days old.
Females: Approximately 63 days old.

Weight range of animals at the start of the study
Males: 321 to 400 g
Females: 208 to 257 g.

Allocation and identification
Allocation
On arrival and non-selective allocation to cages.
On Day 1 of study, all animals were weighed and body weights were reviewed by Study Management before feeding of the treated diets commenced. Absolute body weight of individual animals did not exceed ±20% of the mean for each sex.

Identification of animals
Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages
Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal housing, diet and water supply
Environmental control
Rodent facility
Full barrier - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.

Air supply
Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity
Monitored and maintained within the range of 19-23ºC and 40-70%.
There were no deviations from these ranges.

Lighting
Artificial lighting, 12 hours light : 12 hours dark.

Animal accommodation
Cages
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods.
Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

Cage distribution
The cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups.

Bedding
Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Pre-pairing: up to five males or up to five females
Pairing: one male and one female
Males after mating: up to five males
Gestation: one female
Lactation: one female + litter

Electricity supply
Public supply with automatic stand-by generators.

Environmental enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and from Day 20 after mating to Day 6 of lactation inclusive) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and from Day 20 after mating to Day 6 of lactation inclusive) and replaced when necessary.

Diet supply
Diet: SDS VRF1 Certified powdered diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability
Non-restricted (removed overnight before blood sampling for haematology and blood chemistry investigations).

Water supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability: Non-restricted.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Vehicle
The vehicle was SDS VRF1 certified powdered diet.

Administration
Treatment was restricted to the F0 generation. Animals of the F1 generation received no direct treatment.
Route: Oral, via the diet.
Treated at: Constant dietary concentrations (ppm) for each group.
Control (Group 1): Untreated diet of the same batch.
Frequency: Continuously.
Storage of diet: Due to the volatility risk, diet preparations were stored in amber glass jars. The diets were stored frozen (nominally -20°C) until required for feeding.
Due to the limited ambient stability of the treated diets, fresh diet was supplied twice each week. This ensured that diets were not available to the animals outside of the stability limits, diets were removed from the freezer on the morning of use and allowed to thaw for at least one hour before feeding. The subsequent end-feeder procedures were conducted as early as possible in the morning to ensure that the diet stability was not exceeded. It was accepted that by employing this procedure the animals may have been without food for approximately one and a half hours.

Diet: A record of the usage of the diets was maintained on all occasions when food consumption was measured. This was performed using the initial weight of the diet container and an on-line data check on completion of the feeding procedure to ensure that all cages were fed the correct amount of diet. No significant discrepancy was found.
Details on mating procedure:
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis
Stability and homogeneity
The homogeneity and stability of FRET 08-0338 in laboratory diet for rodents was investigated at concentrations of 100 ppm and 15000 ppm. These investigations demonstrated that FRET 08-0338 was stable in the diet at 100 ppm for four days following ambient storage (nominally 21°C) and for 22 days following frozen storage (nominally -20°C) when stored in amber glass jars. At 15000 ppm, diet formulations were shown to be stable for 22 days following both ambient and frozen storage when stored in amber glass jars.

Achieved concentration
Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance.

Analytical procedure
Apparatus and instrumentation
Gas Chromatograph (GC) Shimadzu GC-2010 Plus Gas Chromatograph
Balances fitted with printers: Capability of weighing to 2, 5 or 6 decimal places
General laboratory apparatus and glassware.

Reagents
Diet: SDS VRF1 certified diet
Acetone: Glass distilled or HPLC grade
Dimethyl phthalate: >99%
Diluent: Acetone (100%)
Stock internal standard: 50 μL of dimethyl phthalate in 200 mL acetone, ca 250 μg/mL

Preparation of standards
A primary standard solution (500 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of FRET 08-0338 in acetone (100 mL). Solutions for instrument calibration were prepared by appropriate dilution of the primary standard using acetone and contained FRET 08-0338 at nominal concentrations of 5 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL and 50 μg/mL.
A volume of internal standard (100 μL) was added to each vial containing 1000 μL of calibration solution prior to injection.
Calibration solutions were injected onto the GC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Sample process
A representative sub-sample (10 g ± 0.5 g) of test diet formulation was extracted (mechanical shaking, 30 minutes) using acetone (100 mL). The extract was clarified by centrifugation (4000 rpm, 10 minutes) and then diluted using acetone as appropriate, to provide a solution containing FRET 08-0338 at an expected concentration between 10 μg/mL and 40 μg/mL.
A volume of internal standard (100 μL) was added to each vial containing 1000 μL of sample solution prior to injection.

The concentration of FRET 08-0338 in the final solution was quantified by GC using Flame Ionisation Detection (FID) detection as detailed in the chromatographic section.

Procedural recoveries
At each analytical occasion procedural recoveries were prepared to cover the range of inclusion levels examined and analysed concurrently with test formulations.

Procedural recoveries were prepared by fortifying samples (10 g ± 0.5 g) of control diet with known amounts of FRET 08-0338. The prepared procedural recoveries were analysed in accordance with the analytical procedure. Procedural recovery values were used to correct sample values.
A volume of internal standard (100 μL) was added to each vial containing 1000 μL of recovery solution prior to injection.

Typical gas chromatographic conditions
Column: ZB-50, 30 m × 0.32 mm ID, 0.25 μm film thickness
Injection mode: Split
Injector temperature: 325°C
Injection volume: 1 µL
Detector type: Flame Ionisation
Detector temperature: 325 °C

Oven temperature programme:
Rate (°C/min) Temperature (°C) Hold time (min)
- 80 1
15 215 0
30 290 1.5

Total run time: 14 minutes
Approximate retention time:
FRET 08-0338 Peak 1: 7.9 minutes
FRET 08-0338 Peak 2: 8.0 minutes
Internal Standard: 9.4 minutes

Gases:
Carrier: Helium at 1.83 mL/min
Split vent: split ratio 1:10
Make up: Helium at 30 mL/min
Oxidant: Air at 400 mL/min
Fuel: Hydrogen at 40 mL/min

The GC system was calibrated using external standards. Peak area data acquired by the data capture software using a 1st order fit was subjected to least squares regression analysis.

Calculation
The sum of the peak area responses of FRET 08-0338 (Peak 1 and 2) and the internal standard in each calibration chromatogram was measured and the peak area response ratio calculated. Calibration curves were constructed by response ratio versus standard concentration. The response ratio for FRET 08-0338 in sample and procedural recovery chromatograms was measured. The concentration of FRET 08-0338 was determined using the following equation:

Analysed concentration (ppm) = ((Y - 1) / S) x (V / W)

Procedural recovery values were determined using the following equation:

Procedural recovery = (analysed concentration (ppm) / Fortified concentration (ppm)) x 100

Sample concentrations were corrected for the overall mean procedural recovery value at analysis using the following equation:

Corrected concentration (ppm) = Analysed concentration (ppm) x 1(100 / R)

Where
Y = Peak area sum response ratio for FRET 08-0338 in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution volume of sample (mL)
W = Sample weight (g)
R = Appropriate mean procedural recovery value at analysis

Validation of the analytical procedure
The analytical procedure was validated prior to treatment by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The limit of detection estimated by examination of control vehicle chromatograms in order to calculate a test substance concentration based on a peak height response equivalent to three times baseline noise.
The limit of quantitation estimated by examination of control vehicle chromatograms in order to calculate a test substance concentration based on a peak height response equivalent to ten times baseline noise.
The linearity of detector response over the calibration standard concentration range.

The precision of injection of the lowest and highest concentration calibration standards.
The accuracy and precision of the method, by determining a minimum of six procedural recoveries at nominal concentrations of 100 ppm and 15000 ppm.
The stability of calibration solutions prepared during the study, and kept in refrigerated storage for a minimum of 1 day, and compared to freshly prepared calibration solutions.

Homogeneity and stability in SDS VRF1 certified diet formulations
Prior to treatment, the homogeneity and stability of FRET 08-0338 in SDS VRF1 certified diet formulations was assessed at the lowest and highest concentrations intended for feeding.
Specimen batches of SDS VRF1 certified diet formulation, containing FRET 08-0338 at nominal concentrations of 100 ppm and 15000 ppm were prepared by Pharmacy personnel and randomly sampled in duplicate from the top, middle and bottom of the turbula drum. Seven extra samples were taken from the drum for stability determination.

Trial 1
Homogeneity: At 100 ppm and 15000 ppm duplicate samples were taken from the top, middle and bottom of the treated diet (analysed singly in accordance with the analytical procedure). Furthermore, at 100 ppm, four sample were taken from each level. The mean analysed concentration was taken as the initial stability timepoint.

Stability: The seven extra diets taken for stability determination were stored at ambient temperature (nominally 21°C) for 1, 2, 4, 8, 15 and 22 days and frozen (nominally -20°C) for 22 days following preparation. The Day 8 and 22 samples at ambient temperature and the Day 22 at frozen storage were analysed, and the remaining samples were kept as contingency. Samples were analysed in duplicate in accordance with the analytical procedure, however diet samples for 100 ppm were re-analysed as contingency, in quadruplicate on Day 27 at frozen storage and for 15000 ppm the Day 8 samples were re-analysed as contingency in quadruplicate (Day 8 + 6). These were discarded once results were obtained.

Trial 2 (100 ppm only)
Stability only: Diet samples were analysed immediately on Day 0 in duplicate, and the remainder was retained frozen as contingency. The remaining diet samples were stored at ambient temperature (nominally 21°C) for 1, 2, and 4 days following preparation. Samples were moved to frozen storage after 1 and 2 days at ambient and analyzed on Day 4 all together. The remaining samples were kept in frozen storage as contingency. Samples were analysed in duplicate in accordance with the analytical procedure, except for the Day 4 at ambient was re-analysed after 5 days stored frozen (Day 4+5) and subsampled in quadruplicate. These were discarded once results were obtained.

Concentration in test formulations
For Weeks 1 and 5 of treatment, representative samples of test diet (200 g) were taken from the turbula mixer drum by Pharmacy personnel and submitted for analysis. Each diet sample was sub-sampled (10 g) in duplicate and analysed in accordance with the analytical procedure. Contingency analysis was performed for Group 2 in Week 1 and Week 5 due to the percentage difference from the mean values being outwith the acceptance criteria. The diet samples were analysed in duplicate for Week 1 and in quadruplicate for Week 5, in accordance with the analytical procedure.

Study clarification and unforeseen events
Trial 1: 100 ppm homogeneity was outwith the acceptance criteria on Day 0, and this was re-assessed by taking further samples. This confirmed that the 100 ppm level showed greater variability than at 15000 ppm. However the overall variability was within the CV= <7.5% which meets the acceptance criteria.
The 15000 ppm diet samples that were analysed on Day 8 had atypically high results. These were reanalysed in quadruplicate after 6 days storage in the freezer and confirmed that the original values were due to analytical error. The contingency results are reported.
The diet samples for 100 ppm that were analysed after 22 days at frozen temperature (-20°C) were atypically high. After further investigation it was found that this was due to an analytical error and the samples were analysed as contingency 5 days after Day 22 (effectively 27 Days) and these results are reported.
At the conclusion of Trial 1, ambient temperature stability could not be confirmed for the low level due to results deviating by more than 10% from Day 0. Therefore a second trial was prepared at 100 ppm only.

Trial 2: Day 1, 2 and 4 diet samples were analysed however the calibration standards failed and the results could not be reported. In addition, the Day 4 duplicate samples showed a high percentage difference from the mean value. The reason for the atypical results is likely due to analytical error.The extracts were re-injected in the next assay, with the Day 4 analysed as contingency in quadruplicate. The contingency analysis (for Day 4+5) meet the acceptance criteria and are reported. The original values for Day 4 are for information only.
Week 1: The diet sample for Group 2 was analysed for contingency because the results initially were not corrected for the procedural recovery values, and it showed that Group 2 was outwith the acceptance criteria. However the results table was only corrected once the contingency analysis was performed. When corrected for the procedural recovery value, the original results for Group 2 was found to be within limits. Therefore Group 2 will be reported with the mean of
four results.
Week 5: The individual results for Group 2 were originally had a high percentage difference from the mean value. Contingency samples were analysed, where four further subsamples were taken to ensure greater accuracy of the concentration. Therefore, there are a total of six results for this group. The coefficient of variation is still outwith the acceptance criteria.

Results
Method validation
The analytical procedure was successfully validated for FRET 08-0338 in SDS VRF1 certified diet with respect to the specificity of chromatographic analysis, limit of detection, limit of quantitation, linearity of detector response, system precision, method accuracy and precision. Results are summarised below:

The specificity of the GC assay was demonstrated by the absence of a peak at the characteristic retention time for FRET 08-0338 in the control sample;
The limit of detection was estimated as 1.30 μg/mL using the operating parameters defined in this procedure;
The limit of quantification was estimated as 4.33 μg/mL using the operating parameters defined in this procedure;
Linearity was confirmed over the nominal concentration range 5 μg/mL to 50 μg/mL with a coefficient of determination >0.999;
The precision of injection was <1% for six replicate injections of standard solutions containing FRET 08-0338 at nominal concentrations of 5 μg/mL and 50 μg/mL;
Method accuracy and precision were confirmed and results are:
mean procedural recovery value of 103.6% (CV=2.37%, n=6) was obtained for 100 ppm and 101.9% (CV=0.53%, n=6) was obtained for 15000 ppm.
The overall mean procedural recovery value of 102.8% (CV=1.87%, n=12) was obtained.
Calibration standard stability was assessed and confirmed for up to 2 days refrigerated storage.

Formulation trial
The homogeneity of FRET 08-0338 in SDS VRF1 certified diet formulations was confirmed at nominal concentrations of 100 ppm and 15000 ppm. Each formulation achieved an accuracy within 10% of the nominal concentration and a precision, measured by the coefficient of variation, of <7.5% for 100 ppm and <5% for 15000 ppm.

Trial 1: The stability of FRET 08-0338 in SDS VRF1 certified diet was confirmed for up to 27 days when frozen at the nominal concentration of 100 ppm, and up to 22 days ambient temperature and frozen temperature at the nominal concentration of 15000 ppm. Mean analysed concentration remained within ±10% of the initial time zero value.

Trial 2: Stability was confirmed for up to 4 days at ambient temperature at 100 ppm.
Procedural recovery results during the trials are presented. Results were between 98.2% and 111.1% which is within the acceptable range of 92.8% to 112.8%.

Concentration in dose formulations
The analysed concentrations of FRET 08-0338 in test formulations analysed during the study, associated procedural recovery data and the deviation of mean results from nominal values are presented. The mean concentrations were within applied limits of +10%/-15%, confirming the accuracy of the formulation.
The percentage difference from the mean values remained within 3% except for Group 2 for both occasions, where the coefficient of variation was 6.58% for Week 1 and 13.3% Week 5.
The procedural recovery values were between 92.8% and 104.3% confirming the continued accuracy and precision of the analytical method.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, limit of quantitation, linearity of detector response, precision of injection, method accuracy and precision and standard stability.
Homogeneity was confirmed for FRET 08-0338 in SDS VRF1 certified diet formulations at nominal concentrations of 100 ppm and 15000 ppm:
· Stability was confirmed at ambient temperature for up to 4 days and frozen for up to 27 days for 100 ppm.
· Stability was confirmed for up to 22 days at ambient temperature and frozen for 15000 ppm.
The mean concentrations of FRET 08-0338 in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. The percentage difference from the mean values remained within 3% and a coefficient of variation for four samples <14%.
Duration of treatment / exposure:
Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 7 of lactation.
Frequency of treatment:
Continuous
Details on study schedule:
- Age at mating of the mated animals in the study:
Males: Minimum of 84 days
Females: Minimum of 77 days
Remarks:
Doses / Concentrations:
0, 1500, 4500 and 15000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, plain diet
Details on study design:
Treatment groups and doses
The selected dietary inclusion levels of 0, 1500, 4500 and 15000 ppm were chosen in conjunction with the Sponsor based on the results of a 14-day preliminary toxicity study conducted at these laboratories (Huntingdon Life Sciences Report No. JMN0069).

In that study, dietary levels of 7500, 11000 and 15000 ppm were investigated; the overall achieved doses during Days 1-15 of treatment at 7500, 11000 and 15000 ppm were 510, 702 and 935 mg/kg/day for males and 517, 709 and 965 mg/kg/day for females, respectively. There was no excessive toxicity observed at any dietary inclusion level investigated. Reductions in food consumption were observed during the first 1-3 days of treatment which indicated that the test diets were slightly unpalatable, and resulted in dose-dependent body weight loss during that period at 11000 or 15000 ppm. There was clear improvement however during Days 4-15 of treatment such that food consumption and body weight performance were similar to Control. The liver was identified as a potential target organ, with a slight increase in liver weight in males and females at 11000 or 15000 ppm.

For the current OECD 422 study, the high dietary inclusion level of 15000 ppm was selected; although the body weight effects at 15000 ppm in the preliminary study were initially marked, the short duration of these effects and rapid return to Control levels of food intake and body weight gain were such that there was no evidence to suggest that a dietary level of 15000 ppm was unsuitable for use. This dietary level was expected to result in an achieved dosage of 1000 mg/kg/day (the highest dosage required by the test guidelines to which this study was designed to meet) in both sexes when averaged over the full treatment period. The lowest dietary concentration (1500 ppm) was expected to provide an achieved dosage of 100 mg/kg/day and to be the No Observed Adverse Effect Level and the intermediate dietary concentration (4500 ppm) was selected as it represented the approximate geometric mean of the low and high dietary concentrations and was expected to provide an achieved dose of 300 mg/kg/day.


Correction factor: None.

Diet: SDS VRF1 Certified powdered.

Method of preparation
For each dietary level, the test substance was incorporated into the diet to provide the required concentration by initial preparation of a premix. The amount of test substance required for the premix was added to an equal amount of sieved diet and stirred. An amount of sieved diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with coarse diet. The premix was then mixed using a Turbula mixer for 200 cycles at 16 rpm.

This premix was diluted with further quantities of plain diet to prepare the required concentration. Each formulation was mixed using a Turbula mixer for 200 cycles at 16 rpm.
Due to the volatility risk, diet preparations were stored in amber glass jars.

Frequency of preparation: Weekly.
Storage of formulation: Frozen (nominally -20°C) immediately following preparation until required for feeding. Diets were then removed from frozen storage on the morning of use.
Test substance accounting: Detailed records of compound usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.


Mating
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.


Terminal procedures
All adult 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.


Time of necropsy
F0 males: After Week 6 investigations completed.
F0 females: Day 8 of lactation (after blood sampling).
F1 offspring: Day 7 of age.


Method of kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.
Offspring : Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.
Positive control:
None
Parental animals: Observations and examinations:
Clinical and behavioural observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). 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 acclimatisation period, observations of the animals and their cages were recorded at least once per day.

Detailed physical examination and arena observations
On Day 1 of study (before treatment commenced), during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation, 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 (during the treatment period), by an observer unaware of the experimental group identities. For logistical reasons, “blind” recording was not possible for animals during pairing or for females after mating and during lactation, therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and behaviour, both 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 behaviour.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed on the five lowest numbered surviving males in each group during Week 5 of treatment and on the five lowest numbered lactating females in each group on Day 4-6 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved into individual cages prior to transport to the testing room. The cage labels on these individual cages showed only the study and animal number. 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 as far as possible 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 5 of treatment for males and on Day 4-6 of lactation for females, the motor activity of the five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.

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 all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body weight
The weight of animals was recorded as follows:
F0 males:
On the day that treatment commenced (Week 0), weekly thereafter and on the day of necropsy.

F0 females:
On the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 3, 6, 13 and 20 after mating.
Day 1, 4, and 7 of lactation.
On the day of necropsy.

Food consumption
The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows:
F0 animals
Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
Due to the limited ambient stability of the treated diets, for females after mating food consumption was performed as follows:
Days 0-2, 3-5, 6-8, 9-12, 13-15 and 16-19 after mating
Days 1-3 and 4-6 of lactation
From these records the mean daily consumption per animal (g/animal/week) or (g/animal/day) was calculated for each phase.

Haematology, peripheral blood
Blood samples were collected after overnight withdrawal of food at the following occasions:
Week 6: The five lowest numbered surviving males per group
Day 8 of lactation: The five lowest numbered surviving females per group

Animals were held under light general anaesthesia 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 using a Bayer Advia 120 analyser:
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell haemoglobin (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 (LUC)
Platelet count (Plt)
Morphology:
Anisocytosis
Microcytosis
Macrocytosis
Hypochromasia
Hyperchromasia

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

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.

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

Blood chemistry
Blood samples were collected after overnight withdrawal of food at the following occasions:
Week 6: The five lowest numbered surviving males per group
Day 8 of lactation: The five lowest numbered surviving females per group


Animals were held under light general anaesthesia 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 Analyser in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

Records made during littering phase
Clinical observations:
Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Females
The following were recorded:
Each uterine horn: Number of implantation sites and corpora lutea.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
Litter size
Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.

Sex ratio of each litter
Recorded on Days 1, 4 and 7 of age.

Individual offspring body weights
Days 1, 4 and 7 of age.
Postmortem examinations (parental animals):
Organ weights
For bilateral organs, left and right organs were weighed together, unless specified previously. Requisite organs were weighed for animals killed at scheduled termination.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid.
Eyes: In Davidson’s fluid.
For all animals examined, samples of any abnormal tissues were retained. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region.

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: The five lowest surviving adult males and females in Groups 1 and 4 at scheduled termination.
Liver: The five lowest surviving adult females in Groups 2 and 3 at scheduled termination.
Kidneys: The five lowest surviving adult males in Groups 2 and 3 at scheduled termination.
Abnormalities only: All F0 animals.
Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light microscopy
Tissues preserved for examination were examined as follows:
Five lowest numbered surviving adult males and females in Groups 1 and 4: All specified
Five lowest numbered surviving adult females in Groups 2 and 3: Liver
Five lowest numbered surviving adult males in Groups 2 and 3: Kidneys
All adult animals: Abnormalities only.

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.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Postmortem examinations (offspring):
Offspring
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination: Normal offspring were discarded. Any externally abnormal offspring was also examined internally. Abnormal tissues were retained.
Statistics:
See below
Reproductive indices:
Pre-coital interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.
Mating performance and fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating = (number of animals mating / animals paired) x 100

Conception rate (%) = (number of aniamls achieivng pregnancy / aniamls mated) x 100

Fertility index (%) = (number of animals achieving pregnanxy / animals paired) x 100


Gestation length
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation length (%) = (number of live litters born / number pregnant) x 100
Offspring viability indices:
Litter size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1 and 7 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival indices
The following were calculated for each litter:

Post implantation survival index (%) = (total number of offspring born / total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (nuber of live off spring on Day 1 after littering / total number of offspring born) x 100

Viability index (%) = (number of live offspring on Day 7 / number of offspring on Day 1 after littering) x 100

Group mean values were calculated from individual litter values.

Sex ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1 and 7 of age.

Percentage males = (number of males in litter / total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See results
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See results
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: See results
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Detailed physical examination and arena observation
Throughout the study, there were no signs observed during the detailed physical examination and arena observations that were attributable to the administration of FRET 08-0338.

Sensory reactivity and grip strength
Sensory reactivity and grip strength were considered to be unaffected by FRET 08-0338 at all dietary inclusion levels investigated. It was noted that the forelimb grip strength of males and females given 4500 or 15000 ppm was slightly lower than Control although in the absence of a clear dose response relationship; statistical significance was not attained and in the absence of any effects on the hindlimb grip strength of these males and females, these minor differences from Control were considered to have occurred fortuitously.

Motor activity
Motor activity scores for males and females given FRET 08-0338 were considered to be unaffected by treatment.
The majority of group mean high and low beam activity scores, including total scores, for all treated males and females were slightly high compared with Controls, with occasional scores for individual 6-minute recording periods attaining statistical significance. All scores attained for the treated males and females were within the Historical Control Data (HCD) range for animals of this strain and age, whereas on several occasions the Control scores were below the HCD range. It was therefore considered that that these differences from Control were due to natural variation and atypical Control scores, rather than treatment with FRET 08-0338.

Body weight
For males receiving 15000 ppm, mean body weight gain was slightly lower than Control in Weeks 1, 3, 4 and 5 of treatment, such that overall mean body weight gain was 20% lower than Control; none of the differences from Control attained statistical significance and the differences were predominantly attributable to one male (No. 11) which showed atypically low weight gain during the course of the study. Mean body weight gain of males was considered unaffected by the presence of FRET 08-0338 in the diet at levels at 1500 or 4500 ppm. It was noted that the mean weight gain of all groups of males (including Controls) was slightly low in Week 3 of treatment when compared to the preceding and following study weeks; this was considered to reflect the impact of being paired with the females during that week.
Among females given FRET 08-0338, a clear and dose-dependent effect on body weight performance was apparent during Week 1 of treatment; females given 1500 or 4500 ppm showed a reduction in mean weight gain and females given 15000 ppm recorded mean body weight loss of 5 grams compared to a mean weight gain of 8 grams among Control females during the same period. Thereafter, during Week 2 of treatment, mean body weight gain was unaffected by FRET 08-0338 administration. As a consequence of the effects seen during Week 1, overall mean body weight gain during the 2-week pre-pairing period was statistically significantly lower than Control for females given 4500 or 15000 ppm (69% and 92% lower, respectively).
After mating, females given 15000 ppm showed low mean body weight gain during the majority of the gestation period, particularly between Days 0-3 and Day 6-13 of gestation where differences from Control attained statistical significance. These differences resulted in overall mean weight gain during Days 0-20 of gestation being 22% lower than Controls. Following parturition, these females recorded mean weight loss of 1 gram during Days 1-4 of lactation compared to mean weight gain of 10 grams among Controls; thereafter to Day 7 of lactation mean body weight gain of these females was slightly higher than Control.
The mean body weight performance of females given 1500 or 4500 ppm was considered unaffected by FRET 08-0338 administration during gestation and lactation. It was noted that the weight gain of females given 4500 ppm was low during Days 1-4 of lactation when compared to Controls (2 grams versus 10 grams); statistical significance was not attained for this difference and overall mean body weight gain during Days 1-7 of lactation was essentially similar to Control, therefore no effect of treatment was considered to be inferred.

Food consumption
When compared to Control values, following the commencement of treatment low food intake was apparent during Days 1-4 for males given 15000 ppm (29% lower than Controls). Thereafter, during the remainder of Week 1 and throughout Weeks 2, 4 and 5 mean food intake was similar to or slightly greater than Controls. The mean food intake of males given 1500 or 4500 ppm was unaffected by FRET 08-0338 administration.
At 15000 ppm, females showed markedly low food intake during Days 1-4 of treatment when compared to Control values (44% lower); thereafter during the remainder of the 2-week pre pairing treatment period mean food intake for these females was similar to Controls. After mating, mean food consumption was statistically significantly lower than Control during
Days 0-2 and Days 6-12 of gestation, coinciding with the periods of low mean weight gain among these females. Following parturition, the mean food intake of these females was statistically significantly lower than Controls throughout Days 1-6 of lactation.
Effects on mean food intake among females given 4500 ppm were limited to a slight reduction in consumption during Days 1-4 of the 2-week pre-pairing treatment period. The food intake of females given 1500 ppm was considered unaffected by FRET 08-0338 administration throughout the treatment period.

Haematology
During Week 6 of treatment, males given 15000 ppm showed statistically significantly low total white blood cell counts when compared to concurrent Controls, and below the HCD range (8.77 - 22.71E+09/L at the 5-95% confidence limits). This difference was attributable to statistically significantly low neutrophil, lymphocyte, basophil and large unstained cell counts when compared to concurrent Controls, which were also below the HCD range at the 5 95% confidence limits. Males given 4500 ppm also showed statistically significantly low total white blood cell counts, predominantly due to low lymphocyte counts; all mean and individual values in this group of males were within the HCD range. There were no changes in leucocytic parameters for males given 1500 ppm, and erythrocytic parameters were unaffected by FRET 08 0338 in all groups of treated males.
Haematological investigations conducted for females on Day 8 of lactation did not reveal any treatment-related changes when compared to Controls.

Blood chemistry
Biochemical changes in the plasma at the end of the treatment period (Week 6 for males and Day 8 of lactation for females) revealed the following differences from Control which, unless otherwise stated, were within the HCD range: alanine aminotransferase activity was slightly high in females given 15000 ppm and was above the 5-95% confidence limits HCD range (26-85 U/L); bilirubin concentration were marginally low in males given 15000 ppm; bile acid concentrations were low for males given 4500 or 15000 ppm although there was no dose response; creatinine concentrations were slightly high for males given 15000 ppm but slightly low for all groups of treated females; glucose concentrations were slightly low for all groups of treated males although in the absence of a dose response; cholesterol concentrations were slightly elevated in all groups of treated females with a dose response apparent (ranging from 31% to 140% higher than Control) and values for females given 15000 ppm were above the HCD range (1.28 - 2.88 mmol/L); triglyceride concentrations were high in males given 15000 ppm (88% higher than Control) and in females given 4500 or 15000 ppm (153% and 458% higher than Control, respectively) with the values for females exceeding the HCD range (0.29 1.20 mmol/L); calcium concentrations were marginally increased in females given 15000 ppm (by 9%) and exceeded the HCD range (2.48 - 2.79 mmol/L); total protein and albumin concentrations were slightly low in females given 4500 or 15000 ppm and albumin/globulin ratio was slightly low in all groups of treated females.

Pre-coital interval, mating performance and fertility, gestation length and gestation index
Mating performance was considered unaffected by treatment with FRET 08-0338, with all pairs mating at the first oestrus opportunity.
There was no evidence of dystocia, all females were pregnant and all successfully gave birth to live young, thus fertility was unaffected by treatment.
Gestation length for all females was within the expected range of 22 to 23 days, and gestation index was 100% in all groups.

Organ weights
At scheduled termination in Week 6 for males and on Day 8 of lactation for females, mean adjusted liver weights were increased in a dose related manner from the mid dose onwards in males (12% higher than Control in the mid dose and 30% higher in the high dose, where it reached statistical significance). In females adjusted liver weights were also increased by 12% in the mid dose and 39% in the high dose, when compared to Controls, with statistical significance attained in the high dose group. The adjusted kidney weight and epididymides weight of males given 4500 or 15000 ppm and the adjusted spleen weight of females in these groups were statistically significantly high when compared to Control, although there was no dose response apparent.
For females given 15000 ppm, adjusted brain weights were marginally but statistically significantly high when compared to Controls although there was no effect on absolute brain weights, indicating that this difference was solely due to the slightly lower terminal body weight of these females and no direct effect of treatment was inferred. In addition, mean adjusted uterus/cervix/oviduct weights were slightly low for females given 15000 ppm when compared to Controls; this difference was considered likely to reflect the stage of oestrus at the time of necropsy and was unrelated to FRET 08-0338 administration.

Macropathology
The macroscopic examination performed at scheduled termination revealed no test substance related lesions. The incidence and distribution of all findings were consistent with the common background seen in Crl:CD(SD) rats at these laboratories.

Histopathology
Changes related to treatment with FRET 08-0338 administration were seen in the kidneys (males) and the liver (females).
Dose descriptor:
NOAEL
Effect level:
93 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reproductive performance based on reduction in the number of uterine implantations at 4500 and 15000 ppm.
Dose descriptor:
NOAEL
Effect level:
104 - 197 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reproductive performance based on reduction in the number of uterine implantations at 4500 and 15000 ppm.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See results
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
F1 litter responses
Offspring clinical signs
There were no treatment-related clinical signs seen among the offspring at any dietary inclusion level investigated.

Litter size, sex ratio and survival indices
Among females given 4500 or 15000 ppm, mean implantation counts were slightly, but statistically significantly lower than Control, with 5/10 and 7/10 females having fewer implantation sites than the lowest concurrent Control, and the mean values being outside the HCD range of 15.4 to 17.5 implantation sites. As a consequence, mean litter size in these groups on Day 1 of lactation was lower than Control. The mean number of implantation sites and mean litter size were unaffected at 1500 ppm.
The mean post-partum corpora lutea count recorded on Day 8 of lactation was similar in all groups of females.
Sex ratio, as assessed by the percentage of males present in each litter, and offspring survival from conception to Day 7 of age was unaffected by FRET 08-0338 administration at all dietary inclusion levels investigated.

Offspring body weight
On Day 1 of age, the mean absolute body weight of offspring in litters derived from parent animals given 15000 ppm was 7% and 8% lower than Control for males and females, respectively, with differences attaining statistical significance. The mean body weight gain of these offspring was 30-31% lower than Control throughout Days 1-7 of age, such that mean absolute body weight on Day 7 of age was 20% lower than Control in both sexes.
At 4500 ppm, mean offspring body weights on Day 1 of age were essentially similar to Control. Mean body weight gain between Days 1 and 7 of age was 7-9% lower than Control such that mean absolute body weight on Day 7 of age was 4-5% lower than Control; none of these difference attained statistical significance and no adverse effect of FRET 08-0338 was considered to be inferred.
The body weight and body weight gain of offspring in the 1500 ppm group was considered unaffected by parental treatment with FRET 08-0338.

Offspring macropathology
There were no macroscopic abnormities detected among offspring which died or were killed prior to scheduled termination or at scheduled termination on Day 7 of age that were indicative of an adverse effect of parental treatment with FRET 08-0338.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
274 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Reduction in birth weight and subsequent body weight gain of the offspring derived from parent animals given 15000 ppm
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
319 - 597 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Reduction in birth weight and subsequent body weight gain of the offspring derived from parent animals given 15000 ppm
Reproductive effects observed:
not specified

Formulation analysis

Homogeneity was confirmed for FRET 08-0338 in SDS VRF1 certified diet formulations at nominal concentrations of 100 ppm and 15000 ppm:

  • Stability was confirmed at ambient temperature for up to 4 days and frozen for up to
    27 days for 100 ppm.
  • Stability was confirmed for up to 22 days at ambient temperature and frozen for
    15000 ppm.

The mean concentrations of FRET 08-0338 in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. The percentage difference from the mean values remained within 3% and a coefficient of variation for four samples <14%.

F0 responses

Achieved dose

Mean achieved doses (mg/kg/day) during the study were as follows:

Text Table 1: Achieved doses (mg/kg/day)

 

Males

Females

Dietary Conc. (ppm)

1500

4500

15000

1500

4500

15000

Period

 

 

 

 

 

 

Weeks 1-5 (males)

93

274

941

-

-

-

Females prior to pairing

-

-

-

104

319

957

Females during gestation

-

-

-

116

372

1140

Females during Days 1-7 of lactation

-

-

-

197

579

1805

 

At the highest dietary concentration (15000 ppm) overall average intake was 941 mg/kg/day for the males, 957 mg/kg/day for the females prior to pairing and 1140 mg/kg/day during gestation. During lactation, achieved intake was much higher than in the other study phases, averaging
1805 mg/kg/day, reflecting the increase in female food consumption in response to the increased physiological demands when rearing the litter. Achieved intake in the 1500 and 4500 ppm groups were in proportion to the levels present in the diet.

Histopathology

Changes related to treatment with FRET 08-0338 administration were seen in the kidneys (males) and the liver (females).

Kidney

Slight to moderate hyaline droplets in the cortical tubules was seen in all groups of treated males. Minimal to slight cortical basophilic tubules were seen in males given 4500 or 15000 ppm.

Summary of treatment related findings in the kidney for animals killed after scheduled treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Level (ppm)

0

1500

4500

15000

0

1500

4500

15000

 

 

 

 

 

 

 

 

 

Cortical tubules with hyaline droplets

 

 

 

 

 

 

 

 

Slight

0

5

2

0

0

-

-

0

Moderate

0

1

2

5

0

-

-

0

Total

0

6

4

5

0

-

-

0

 

 

 

 

 

 

 

 

 

 Cortical basophilic tubules

 

 

 

 

 

 

 

 

Minimal

0

0

0

2

0

-

-

0

Slight

0

0

1

1

0

-

-

0

Total

0

0

1

3

0

-

-

0

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

6

5

5

5

0

0

5

 

 

 

 

 

 

 

 

 

 

Liver

Minimal to moderate vacuolation of periportal/diffuse hepatocytes was observed in the liver of females given 4500 or 15000 ppm.

Summary of treatment related findings in the liver after scheduled treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Level (ppm)

0

1500

4500

15000

0

1500

4500

15000

 

 

 

 

 

 

 

 

 

Hepatocyte vacuolation, periportal/diffuse

 

 

 

 

 

 

 

 

Minimal

0

-

-

0

0

0

1

1

Slight

0

-

-

0

0

0

1

1

Moderate

0

-

-

0

0

0

0

1

Total

0

-

-

0

0

0

2

3

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

0

0

5

5

5

5

5

 

 

 

 

 

 

 

 

 

 

Seminiferous tubules were evaluated with respect to their stage in spermatogenic cycle and the integrity of the cell types present within different stages. No cell or stage specific abnormalities were noted.

All other histological changes were considered to be unrelated to treatment.

Conclusions:
Dietary administration of FRET 08-0338 to CD rats for at least 5 weeks in males and approximately 7 weeks in females at concentrations up to and including 15000 ppm was generally well tolerated. Test article related histopathological changes were apparent in the liver of females given 4500 ppm and above, and in the kidneys of males in all treated groups. The liver changes (high values for cholesterol, high values for triglycerides, high adjusted liver weights, elevated alanine aminotransferase activity and hepatocyte vacuolation) observed in females are considered to be related to treatment. These changes may be (partly) due to metabolism of the test substance, and they may also be adverse. The kidney changes detected in the males (hyaline droplets in tubules and cortical basophilic tubules) were consistent with well documented species-specific responses of the male rat in response to the administration with hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. Based on these considerations, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity is based on potentially adverse liver effects and is therefore set at 1500 ppm (equivalent to 93 mg/kg bw/day for males and 104-197 mg/kg bw/day for females).
The reduction in the number of uterine implantations at 4500 and 15000 ppm was minimal (= <15% of Control) and not related to any other changes in reproductive parameters, such as post implantation loss. This effect, however, was statistically significant and outside of the historical control values (representing 10 OECD TG 422 studies). Although this effect may be related to the lower maternal body weight (gain), it is considered to be of uncertain aetiology and therefore potentially adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive performance was 1500 ppm (equivalent to 93 mg/kg bw/day for males and 104-197 mg kg/bw/day for females).
The reduction in birth weight and subsequent body weight gain of the offspring derived from parent animals given 15000 ppm was also of uncertain aetiology, and therefore potentially adverse. It was therefore concluded that the NOAEL for offspring growth, survival and development was 4500 ppm (equivalent to 274 mg/kg bw/day for males and 319 579 mg/kg bw/day for females).
Executive summary:

The reproductive toxicity of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 422 using a screening method. The NOAEL for offspring growth, survival and development was 4500 ppm (equivalent to 274 mg/kg bw/day for males and 319‑579 mg/kg bw/day for females).The NOAEL for reproductive performance was 1500 ppm (equivalent to 93 mg/kg bw/day for males and 104-197 mg kg/bw/day for females).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
104 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information
Short description of key information:
The reproductive toxicity of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 422 using a screening method. The NOAEL for offspring growth, survival and development was 4500 ppm (equivalent to 274 mg/kg bw/day for males and 319‑579 mg/kg bw/day for females).The NOAEL for reproductive performance was 1500 ppm (equivalent to 93 mg/kg bw/day for males and 104-197 mg kg/bw/day for females).

Justification for selection of Effect on fertility via oral route:
The study was conducted on the target substance in vivo, in an appropriate test species and according to internationally recognised guidelines.

Effects on developmental toxicity

Description of key information
The developmental toxicity / teratogenicity of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 422 using a screening method. The NOAEL for offspring growth, survival and development was 4500 ppm (equivalent to 274 mg/kg bw/day for males and 319‑579 mg/kg bw/day for females). The NOAEL for reproductive performance was 1500 ppm (equivalent to 93 mg/kg bw/day for males and 104-197 mg kg/bw/day for females).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 08 June 2015 and 04 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it was conducted according to OECD Test Guideline 422 using a screening method and in compliance with GLP.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 screen for reproductive/ developmental effects
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
CD-1
Details on test animals and environmental conditions:
Strain/Species Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd.
Number of animals ordered: 44 males and 44 females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatisation: Five days before commencement of treatment.
Age of animals at the start of the study
Males: Approximately 70 days old.
Females: Approximately 63 days old.

Weight range of animals at the start of the study
Males: 321 to 400 g
Females: 208 to 257 g

Allocation and identification
Allocation
On arrival and non-selective allocation to cages.
On Day 1 of study, all animals were weighed and body weights were reviewed by Study Management before feeding of the treated diets commenced. Absolute body weight of individual animals did not exceed ±20% of the mean for each sex.

Identification of animals
Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages
Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal housing, diet and water supply
Environmental control
Rodent facility
Full barrier - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.

Air supply
Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity
Monitored and maintained within the range of 19-23ºC and 40-70%.
There were no deviations from these ranges.

Lighting
Artificial lighting, 12 hours light : 12 hours dark.

Animal accommodation
Cages
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods.
Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

Cage distribution
The cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups.

Bedding
Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Pre-pairing: up to five males or up to five females
Pairing: one male and one female
Males after mating: up to five males
Gestation: one female
Lactation: one female + litter

Electricity supply
Public supply with automatic stand-by generators.

Environmental enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and from Day 20 after mating to Day 6 of lactation inclusive) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and from Day 20 after mating to Day 6 of lactation inclusive) and replaced when necessary.

Diet supply
Diet: SDS VRF1 Certified powdered diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability
Non-restricted (removed overnight before blood sampling for haematology and blood chemistry investigations).

Water supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability: Non-restricted.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Vehicle
The vehicle was SDS VRF1 certified powdered diet.

Administration
Treatment was restricted to the F0 generation. Animals of the F1 generation received no direct treatment.
Route: Oral, via the diet.
Treated at: Constant dietary concentrations (ppm) for each group.
Control (Group 1): Untreated diet of the same batch.
Frequency: Continuously.
Storage of diet: Due to the volatility risk, diet preparations were stored in amber glass jars. The diets were stored frozen (nominally -20°C) until required for feeding.
Due to the limited ambient stability of the treated diets, fresh diet was supplied twice each week. This ensured that diets were not available to the animals outside of the stability limits, diets were removed from the freezer on the morning of use and allowed to thaw for at least one hour before feeding. The subsequent end-feeder procedures were conducted as early as possible in the morning to ensure that the diet stability was not exceeded. It was accepted that by employing this procedure the animals may have been without food for approximately one and a half hours.

Diet: A record of the usage of the diets was maintained on all occasions when food consumption was measured. This was performed using the initial weight of the diet container and an on-line data check on completion of the feeding procedure to ensure that all cages were fed the correct amount of diet. No significant discrepancy was found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis
Stability and homogeneity
The homogeneity and stability of FRET 08-0338 in laboratory diet for rodents was investigated at concentrations of 100 ppm and 15000 ppm. These investigations demonstrated that FRET 08-0338 was stable in the diet at 100 ppm for four days following ambient storage (nominally 21°C) and for 22 days following frozen storage (nominally -20°C) when stored in amber glass jars. At 15000 ppm, diet formulations were shown to be stable for 22 days following both ambient and frozen storage when stored in amber glass jars.

Achieved concentration
Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance.

Analytical procedure
Apparatus and instrumentation
Gas Chromatograph (GC) Shimadzu GC-2010 Plus Gas Chromatograph
Balances fitted with printers: Capability of weighing to 2, 5 or 6 decimal places
General laboratory apparatus and glassware.

Reagents
Diet: SDS VRF1 certified diet
Acetone: Glass distilled or HPLC grade
Dimethyl phthalate: >99%
Diluent: Acetone (100%)
Stock internal standard: 50 μL of dimethyl phthalate in 200 mL acetone, ca 250 μg/mL

Preparation of standards
A primary standard solution (500 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of FRET 08-0338 in acetone (100 mL). Solutions for instrument calibration were prepared by appropriate dilution of the primary standard using acetone and contained FRET 08-0338 at nominal concentrations of 5 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL and 50 μg/mL.
A volume of internal standard (100 μL) was added to each vial containing 1000 μL of calibration solution prior to injection.
Calibration solutions were injected onto the GC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.

Sample process
A representative sub-sample (10 g ± 0.5 g) of test diet formulation was extracted (mechanical shaking, 30 minutes) using acetone (100 mL). The extract was clarified by centrifugation (4000 rpm, 10 minutes) and then diluted using acetone as appropriate, to provide a solution containing FRET 08-0338 at an expected concentration between 10 μg/mL and 40 μg/mL.
A volume of internal standard (100 μL) was added to each vial containing 1000 μL of sample solution prior to injection.

The concentration of FRET 08-0338 in the final solution was quantified by GC using Flame Ionisation Detection (FID) detection as detailed in the chromatographic section.

Procedural recoveries
At each analytical occasion procedural recoveries were prepared to cover the range of inclusion levels examined and analysed concurrently with test formulations.

Procedural recoveries were prepared by fortifying samples (10 g ± 0.5 g) of control diet with known amounts of FRET 08-0338. The prepared procedural recoveries were analysed in accordance with the analytical procedure. Procedural recovery values were used to correct sample values.
A volume of internal standard (100 μL) was added to each vial containing 1000 μL of recovery solution prior to injection.

Typical gas chromatographic conditions
Column: ZB-50, 30 m × 0.32 mm ID, 0.25 μm film thickness
Injection mode: Split
Injector temperature: 325°C
Injection volume: 1 µL
Detector type: Flame Ionisation
Detector temperature: 325 °C

Oven temperature programme:
Rate (°C/min) Temperature (°C) Hold time (min)
- 80 1
15 215 0
30 290 1.5

Total run time: 14 minutes
Approximate retention time:
FRET 08-0338 Peak 1: 7.9 minutes
FRET 08-0338 Peak 2: 8.0 minutes
Internal Standard: 9.4 minutes

Gases:
Carrier: Helium at 1.83 mL/min
Split vent: split ratio 1:10
Make up: Helium at 30 mL/min
Oxidant: Air at 400 mL/min
Fuel: Hydrogen at 40 mL/min

The GC system was calibrated using external standards. Peak area data acquired by the data capture software using a 1st order fit was subjected to least squares regression analysis.

Calculation
The sum of the peak area responses of FRET 08-0338 (Peak 1 and 2) and the internal standard in each calibration chromatogram was measured and the peak area response ratio calculated. Calibration curves were constructed by response ratio versus standard concentration. The response ratio for FRET 08-0338 in sample and procedural recovery chromatograms was measured. The concentration of FRET 08-0338 was determined using the following equation:

Analysed concentration (ppm) = ((Y - 1) / S) x (V / W)

Procedural recovery values were determined using the following equation:

Procedural recovery = (analysed concentration (ppm) / Fortified concentration (ppm)) x 100

Sample concentrations were corrected for the overall mean procedural recovery value at analysis using the following equation:

Corrected concentration (ppm) = Analysed concentration (ppm) x 1(100 / R)

Where
Y = Peak area sum response ratio for FRET 08-0338 in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution volume of sample (mL)
W = Sample weight (g)
R = Appropriate mean procedural recovery value at analysis

Validation of the analytical procedure
The analytical procedure was validated prior to treatment by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The limit of detection estimated by examination of control vehicle chromatograms in order to calculate a test substance concentration based on a peak height response equivalent to three times baseline noise.
The limit of quantitation estimated by examination of control vehicle chromatograms in order to calculate a test substance concentration based on a peak height response equivalent to ten times baseline noise.
The linearity of detector response over the calibration standard concentration range.

The precision of injection of the lowest and highest concentration calibration standards.
The accuracy and precision of the method, by determining a minimum of six procedural recoveries at nominal concentrations of 100 ppm and 15000 ppm.
The stability of calibration solutions prepared during the study, and kept in refrigerated storage for a minimum of 1 day, and compared to freshly prepared calibration solutions.

Homogeneity and stability in SDS VRF1 certified diet formulations
Prior to treatment, the homogeneity and stability of FRET 08-0338 in SDS VRF1 certified diet formulations was assessed at the lowest and highest concentrations intended for feeding.
Specimen batches of SDS VRF1 certified diet formulation, containing FRET 08-0338 at nominal concentrations of 100 ppm and 15000 ppm were prepared by Pharmacy personnel and randomly sampled in duplicate from the top, middle and bottom of the turbula drum. Seven extra samples were taken from the drum for stability determination.

Trial 1
Homogeneity: At 100 ppm and 15000 ppm duplicate samples were taken from the top, middle and bottom of the treated diet (analysed singly in accordance with the analytical procedure). Furthermore, at 100 ppm, four sample were taken from each level. The mean analysed concentration was taken as the initial stability timepoint.

Stability: The seven extra diets taken for stability determination were stored at ambient temperature (nominally 21°C) for 1, 2, 4, 8, 15 and 22 days and frozen (nominally -20°C) for 22 days following preparation. The Day 8 and 22 samples at ambient temperature and the Day 22 at frozen storage were analysed, and the remaining samples were kept as contingency. Samples were analysed in duplicate in accordance with the analytical procedure, however diet samples for 100 ppm were re-analysed as contingency, in quadruplicate on Day 27 at frozen storage and for 15000 ppm the Day 8 samples were re-analysed as contingency in quadruplicate (Day 8 + 6). These were discarded once results were obtained.

Trial 2 (100 ppm only)
Stability only: Diet samples were analysed immediately on Day 0 in duplicate, and the remainder was retained frozen as contingency. The remaining diet samples were stored at ambient temperature (nominally 21°C) for 1, 2, and 4 days following preparation. Samples were moved to frozen storage after 1 and 2 days at ambient and analyzed on Day 4 all together. The remaining samples were kept in frozen storage as contingency. Samples were analysed in duplicate in accordance with the analytical procedure, except for the Day 4 at ambient was re-analysed after 5 days stored frozen (Day 4+5) and subsampled in quadruplicate. These were discarded once results were obtained.

Concentration in test formulations
For Weeks 1 and 5 of treatment, representative samples of test diet (200 g) were taken from the turbula mixer drum by Pharmacy personnel and submitted for analysis. Each diet sample was sub-sampled (10 g) in duplicate and analysed in accordance with the analytical procedure. Contingency analysis was performed for Group 2 in Week 1 and Week 5 due to the percentage difference from the mean values being outwith the acceptance criteria. The diet samples were analysed in duplicate for Week 1 and in quadruplicate for Week 5, in accordance with the analytical procedure.

Study clarification and unforeseen events
Trial 1: 100 ppm homogeneity was outwith the acceptance criteria on Day 0, and this was re-assessed by taking further samples. This confirmed that the 100 ppm level showed greater variability than at 15000 ppm. However the overall variability was within the CV= <7.5% which meets the acceptance criteria.
The 15000 ppm diet samples that were analysed on Day 8 had atypically high results. These were reanalysed in quadruplicate after 6 days storage in the freezer and confirmed that the original values were due to analytical error. The contingency results are reported.
The diet samples for 100 ppm that were analysed after 22 days at frozen temperature (-20°C) were atypically high. After further investigation it was found that this was due to an analytical error and the samples were analysed as contingency 5 days after Day 22 (effectively 27 Days) and these results are reported.
At the conclusion of Trial 1, ambient temperature stability could not be confirmed for the low level due to results deviating by more than 10% from Day 0. Therefore a second trial was prepared at 100 ppm only.

Trial 2: Day 1, 2 and 4 diet samples were analysed however the calibration standards failed and the results could not be reported. In addition, the Day 4 duplicate samples showed a high percentage difference from the mean value. The reason for the atypical results is likely due to analytical error.The extracts were re-injected in the next assay, with the Day 4 analysed as contingency in quadruplicate. The contingency analysis (for Day 4+5) meet the acceptance criteria and are reported. The original values for Day 4 are for information only.
Week 1: The diet sample for Group 2 was analysed for contingency because the results initially were not corrected for the procedural recovery values, and it showed that Group 2 was outwith the acceptance criteria. However the results table was only corrected once the contingency analysis was performed. When corrected for the procedural recovery value, the original results for Group 2 was found to be within limits. Therefore Group 2 will be reported with the mean of
four results.
Week 5: The individual results for Group 2 were originally had a high percentage difference from the mean value. Contingency samples were analysed, where four further subsamples were taken to ensure greater accuracy of the concentration. Therefore, there are a total of six results for this group. The coefficient of variation is still outwith the acceptance criteria.

Results
Method validation
The analytical procedure was successfully validated for FRET 08-0338 in SDS VRF1 certified diet with respect to the specificity of chromatographic analysis, limit of detection, limit of quantitation, linearity of detector response, system precision, method accuracy and precision. Results are summarised below:

The specificity of the GC assay was demonstrated by the absence of a peak at the characteristic retention time for FRET 08-0338 in the control sample;
The limit of detection was estimated as 1.30 μg/mL using the operating parameters defined in this procedure;
The limit of quantification was estimated as 4.33 μg/mL using the operating parameters defined in this procedure;
Linearity was confirmed over the nominal concentration range 5 μg/mL to 50 μg/mL with a coefficient of determination >0.999;
The precision of injection was <1% for six replicate injections of standard solutions containing FRET 08-0338 at nominal concentrations of 5 μg/mL and 50 μg/mL;
Method accuracy and precision were confirmed and results are:
mean procedural recovery value of 103.6% (CV=2.37%, n=6) was obtained for 100 ppm and 101.9% (CV=0.53%, n=6) was obtained for 15000 ppm.
The overall mean procedural recovery value of 102.8% (CV=1.87%, n=12) was obtained.
Calibration standard stability was assessed and confirmed for up to 2 days refrigerated storage.

Formulation trial
The homogeneity of FRET 08-0338 in SDS VRF1 certified diet formulations was confirmed at nominal concentrations of 100 ppm and 15000 ppm. Each formulation achieved an accuracy within 10% of the nominal concentration and a precision, measured by the coefficient of variation, of <7.5% for 100 ppm and <5% for 15000 ppm.

Trial 1: The stability of FRET 08-0338 in SDS VRF1 certified diet was confirmed for up to 27 days when frozen at the nominal concentration of 100 ppm, and up to 22 days ambient temperature and frozen temperature at the nominal concentration of 15000 ppm. Mean analysed concentration remained within ±10% of the initial time zero value.

Trial 2: Stability was confirmed for up to 4 days at ambient temperature at 100 ppm.
Procedural recovery results during the trials are presented. Results were between 98.2% and 111.1% which is within the acceptable range of 92.8% to 112.8%.

Concentration in dose formulations
The analysed concentrations of FRET 08-0338 in test formulations analysed during the study, associated procedural recovery data and the deviation of mean results from nominal values are presented. The mean concentrations were within applied limits of +10%/-15%, confirming the accuracy of the formulation.
The percentage difference from the mean values remained within 3% except for Group 2 for both occasions, where the coefficient of variation was 6.58% for Week 1 and 13.3% Week 5.
The procedural recovery values were between 92.8% and 104.3% confirming the continued accuracy and precision of the analytical method.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, limit of quantitation, linearity of detector response, precision of injection, method accuracy and precision and standard stability.
Homogeneity was confirmed for FRET 08-0338 in SDS VRF1 certified diet formulations at nominal concentrations of 100 ppm and 15000 ppm:
· Stability was confirmed at ambient temperature for up to 4 days and frozen for up to 27 days for 100 ppm.
· Stability was confirmed for up to 22 days at ambient temperature and frozen for 15000 ppm.
The mean concentrations of FRET 08-0338 in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. The percentage difference from the mean values remained within 3% and a coefficient of variation for four samples <14%.
Details on mating procedure:
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Duration of treatment / exposure:
Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 7 of lactation.
Frequency of treatment:
Continuous
Duration of test:
Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 7 of lactation.
Remarks:
Doses / Concentrations:
0, 1500, 4500 and 15000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, plain diet
Details on study design:
Treatment groups and doses
The selected dietary inclusion levels of 0, 1500, 4500 and 15000 ppm were chosen in conjunction with the Sponsor based on the results of a 14-day preliminary toxicity study conducted at these laboratories (Huntingdon Life Sciences Report No. JMN0069).

In that study, dietary levels of 7500, 11000 and 15000 ppm were investigated; the overall achieved doses during Days 1-15 of treatment at 7500, 11000 and 15000 ppm were 510, 702 and 935 mg/kg/day for males and 517, 709 and 965 mg/kg/day for females, respectively. There was no excessive toxicity observed at any dietary inclusion level investigated. Reductions in food consumption were observed during the first 1-3 days of treatment which indicated that the test diets were slightly unpalatable, and resulted in dose-dependent body weight loss during that period at 11000 or 15000 ppm. There was clear improvement however during Days 4-15 of treatment such that food consumption and body weight performance were similar to Control. The liver was identified as a potential target organ, with a slight increase in liver weight in males and females at 11000 or 15000 ppm.

For the current OECD 422 study, the high dietary inclusion level of 15000 ppm was selected; although the body weight effects at 15000 ppm in the preliminary study were initially marked, the short duration of these effects and rapid return to Control levels of food intake and body weight gain were such that there was no evidence to suggest that a dietary level of 15000 ppm was unsuitable for use. This dietary level was expected to result in an achieved dosage of 1000 mg/kg/day (the highest dosage required by the test guidelines to which this study was designed to meet) in both sexes when averaged over the full treatment period. The lowest dietary concentration (1500 ppm) was expected to provide an achieved dosage of 100 mg/kg/day and to be the No Observed Adverse Effect Level and the intermediate dietary concentration (4500 ppm) was selected as it represented the approximate geometric mean of the low and high dietary concentrations and was expected to provide an achieved dose of 300 mg/kg/day.

Correction factor: None.

Diet: SDS VRF1 Certified powdered.

Method of preparation
For each dietary level, the test substance was incorporated into the diet to provide the required concentration by initial preparation of a premix. The amount of test substance required for the premix was added to an equal amount of sieved diet and stirred. An amount of sieved diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with coarse diet. The premix was then mixed using a Turbula mixer for 200 cycles at 16 rpm.

This premix was diluted with further quantities of plain diet to prepare the required concentration. Each formulation was mixed using a Turbula mixer for 200 cycles at 16 rpm.
Due to the volatility risk, diet preparations were stored in amber glass jars.

Frequency of preparation: Weekly.
Storage of formulation: Frozen (nominally -20°C) immediately following preparation until required for feeding. Diets were then removed from frozen storage on the morning of use.
Test substance accounting: Detailed records of compound usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.


Mating
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.


Terminal procedures
All adult 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.


Time of necropsy
F0 males: After Week 6 investigations completed.
F0 females: Day 8 of lactation (after blood sampling).
F1 offspring: Day 7 of age.


Method of kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.
Offspring : Intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.
Maternal examinations:
Clinical and behavioural observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). 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 acclimatisation period, observations of the animals and their cages were recorded at least once per day.

Detailed physical examination and arena observations
On Day 1 of study (before treatment commenced), during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation, 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 (during the treatment period), by an observer unaware of the experimental group identities. For logistical reasons, “blind” recording was not possible for animals during pairing or for females after mating and during lactation, therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and behaviour, both 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 behaviour.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed on the five lowest numbered surviving males in each group during Week 5 of treatment and on the five lowest numbered lactating females in each group on Day 4-6 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved into individual cages prior to transport to the testing room. The cage labels on these individual cages showed only the study and animal number. 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 as far as possible 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 5 of treatment for males and on Day 4-6 of lactation for females, the motor activity of the five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.

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 all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body weight
The weight of animals was recorded as follows:
F0 males:
On the day that treatment commenced (Week 0), weekly thereafter and on the day of necropsy.

F0 females:
On the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 3, 6, 13 and 20 after mating.
Day 1, 4, and 7 of lactation.
On the day of necropsy.

Food consumption
The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows:
F0 animals
Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
Due to the limited ambient stability of the treated diets, for females after mating food consumption was performed as follows:
Days 0-2, 3-5, 6-8, 9-12, 13-15 and 16-19 after mating
Days 1-3 and 4-6 of lactation
From these records the mean daily consumption per animal (g/animal/week) or (g/animal/day) was calculated for each phase.

Haematology, peripheral blood
Blood samples were collected after overnight withdrawal of food at the following occasions:
Week 6: The five lowest numbered surviving males per group
Day 8 of lactation: The five lowest numbered surviving females per group

Animals were held under light general anaesthesia 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 using a Bayer Advia 120 analyser:
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell haemoglobin (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 (LUC)
Platelet count (Plt)
Morphology:
Anisocytosis
Microcytosis
Macrocytosis
Hypochromasia
Hyperchromasia

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

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.

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

Blood chemistry
Blood samples were collected after overnight withdrawal of food at the following occasions:
Week 6: The five lowest numbered surviving males per group
Day 8 of lactation: The five lowest numbered surviving females per group


Animals were held under light general anaesthesia 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 Analyser in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

Records made during littering phase
Clinical observations:
Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Ovaries and uterine content:
The following were recorded:
Each uterine horn: Number of implantation sites and corpora lutea.
Fetal examinations:
Offspring
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination: Normal offspring were discarded. Any externally abnormal offspring was also examined internally. Abnormal tissues were retained.
Statistics:
See below
Indices:
See below
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: See results

Details on maternal toxic effects:
Detailed physical examination and arena observation
Throughout the study, there were no signs observed during the detailed physical examination and arena observations that were attributable to the administration of FRET 08-0338.

Sensory reactivity and grip strength
Sensory reactivity and grip strength were considered to be unaffected by FRET 08-0338 at all dietary inclusion levels investigated. It was noted that the forelimb grip strength of males and females given 4500 or 15000 ppm was slightly lower than Control although in the absence of a clear dose response relationship; statistical significance was not attained and in the absence of any effects on the hindlimb grip strength of these males and females, these minor differences from Control were considered to have occurred fortuitously.

Motor activity
Motor activity scores for males and females given FRET 08-0338 were considered to be unaffected by treatment.
The majority of group mean high and low beam activity scores, including total scores, for all treated males and females were slightly high compared with Controls, with occasional scores for individual 6-minute recording periods attaining statistical significance. All scores attained for the treated males and females were within the Historical Control Data (HCD) range for animals of this strain and age, whereas on several occasions the Control scores were below the HCD range. It was therefore considered that that these differences from Control were due to natural variation and atypical Control scores, rather than treatment with FRET 08-0338.

Body weight
For males receiving 15000 ppm, mean body weight gain was slightly lower than Control in Weeks 1, 3, 4 and 5 of treatment, such that overall mean body weight gain was 20% lower than Control; none of the differences from Control attained statistical significance and the differences were predominantly attributable to one male (No. 11) which showed atypically low weight gain during the course of the study. Mean body weight gain of males was considered unaffected by the presence of FRET 08-0338 in the diet at levels at 1500 or 4500 ppm. It was noted that the mean weight gain of all groups of males (including Controls) was slightly low in Week 3 of treatment when compared to the preceding and following study weeks; this was considered to reflect the impact of being paired with the females during that week.
Among females given FRET 08-0338, a clear and dose-dependent effect on body weight performance was apparent during Week 1 of treatment; females given 1500 or 4500 ppm showed a reduction in mean weight gain and females given 15000 ppm recorded mean body weight loss of 5 grams compared to a mean weight gain of 8 grams among Control females during the same period. Thereafter, during Week 2 of treatment, mean body weight gain was unaffected by FRET 08-0338 administration. As a consequence of the effects seen during Week 1, overall mean body weight gain during the 2-week pre-pairing period was statistically significantly lower than Control for females given 4500 or 15000 ppm (69% and 92% lower, respectively).
After mating, females given 15000 ppm showed low mean body weight gain during the majority of the gestation period, particularly between Days 0-3 and Day 6-13 of gestation where differences from Control attained statistical significance. These differences resulted in overall mean weight gain during Days 0-20 of gestation being 22% lower than Controls. Following parturition, these females recorded mean weight loss of 1 gram during Days 1-4 of lactation compared to mean weight gain of 10 grams among Controls; thereafter to Day 7 of lactation mean body weight gain of these females was slightly higher than Control.
The mean body weight performance of females given 1500 or 4500 ppm was considered unaffected by FRET 08-0338 administration during gestation and lactation. It was noted that the weight gain of females given 4500 ppm was low during Days 1-4 of lactation when compared to Controls (2 grams versus 10 grams); statistical significance was not attained for this difference and overall mean body weight gain during Days 1-7 of lactation was essentially similar to Control, therefore no effect of treatment was considered to be inferred.

Food consumption
When compared to Control values, following the commencement of treatment low food intake was apparent during Days 1-4 for males given 15000 ppm (29% lower than Controls). Thereafter, during the remainder of Week 1 and throughout Weeks 2, 4 and 5 mean food intake was similar to or slightly greater than Controls. The mean food intake of males given 1500 or 4500 ppm was unaffected by FRET 08-0338 administration.
At 15000 ppm, females showed markedly low food intake during Days 1-4 of treatment when compared to Control values (44% lower); thereafter during the remainder of the 2-week pre pairing treatment period mean food intake for these females was similar to Controls. After mating, mean food consumption was statistically significantly lower than Control during
Days 0-2 and Days 6-12 of gestation, coinciding with the periods of low mean weight gain among these females. Following parturition, the mean food intake of these females was statistically significantly lower than Controls throughout Days 1-6 of lactation.
Effects on mean food intake among females given 4500 ppm were limited to a slight reduction in consumption during Days 1-4 of the 2-week pre-pairing treatment period. The food intake of females given 1500 ppm was considered unaffected by FRET 08-0338 administration throughout the treatment period.

Haematology
During Week 6 of treatment, males given 15000 ppm showed statistically significantly low total white blood cell counts when compared to concurrent Controls, and below the HCD range (8.77 - 22.71E+09/L at the 5-95% confidence limits). This difference was attributable to statistically significantly low neutrophil, lymphocyte, basophil and large unstained cell counts when compared to concurrent Controls, which were also below the HCD range at the 5 95% confidence limits. Males given 4500 ppm also showed statistically significantly low total white blood cell counts, predominantly due to low lymphocyte counts; all mean and individual values in this group of males were within the HCD range. There were no changes in leucocytic parameters for males given 1500 ppm, and erythrocytic parameters were unaffected by FRET 08 0338 in all groups of treated males.
Haematological investigations conducted for females on Day 8 of lactation did not reveal any treatment-related changes when compared to Controls.

Blood chemistry
Biochemical changes in the plasma at the end of the treatment period (Week 6 for males and Day 8 of lactation for females) revealed the following differences from Control which, unless otherwise stated, were within the HCD range: alanine aminotransferase activity was slightly high in females given 15000 ppm and was above the 5-95% confidence limits HCD range (26-85 U/L); bilirubin concentration were marginally low in males given 15000 ppm; bile acid concentrations were low for males given 4500 or 15000 ppm although there was no dose response; creatinine concentrations were slightly high for males given 15000 ppm but slightly low for all groups of treated females; glucose concentrations were slightly low for all groups of treated males although in the absence of a dose response; cholesterol concentrations were slightly elevated in all groups of treated females with a dose response apparent (ranging from 31% to 140% higher than Control) and values for females given 15000 ppm were above the HCD range (1.28 - 2.88 mmol/L); triglyceride concentrations were high in males given 15000 ppm (88% higher than Control) and in females given 4500 or 15000 ppm (153% and 458% higher than Control, respectively) with the values for females exceeding the HCD range (0.29 1.20 mmol/L); calcium concentrations were marginally increased in females given 15000 ppm (by 9%) and exceeded the HCD range (2.48 - 2.79 mmol/L); total protein and albumin concentrations were slightly low in females given 4500 or 15000 ppm and albumin/globulin ratio was slightly low in all groups of treated females.

Pre-coital interval, mating performance and fertility, gestation length and gestation index
Mating performance was considered unaffected by treatment with FRET 08-0338, with all pairs mating at the first oestrus opportunity.
There was no evidence of dystocia, all females were pregnant and all successfully gave birth to live young, thus fertility was unaffected by treatment.
Gestation length for all females was within the expected range of 22 to 23 days, and gestation index was 100% in all groups.

Organ weights
At scheduled termination in Week 6 for males and on Day 8 of lactation for females, mean adjusted liver weights were increased in a dose related manner from the mid dose onwards in males (12% higher than Control in the mid dose and 30% higher in the high dose, where it reached statistical significance). In females adjusted liver weights were also increased by 12% in the mid dose and 39% in the high dose, when compared to Controls, with statistical significance attained in the high dose group. The adjusted kidney weight and epididymides weight of males given 4500 or 15000 ppm and the adjusted spleen weight of females in these groups were statistically significantly high when compared to Control, although there was no dose response apparent.
For females given 15000 ppm, adjusted brain weights were marginally but statistically significantly high when compared to Controls although there was no effect on absolute brain weights, indicating that this difference was solely due to the slightly lower terminal body weight of these females and no direct effect of treatment was inferred. In addition, mean adjusted uterus/cervix/oviduct weights were slightly low for females given 15000 ppm when compared to Controls; this difference was considered likely to reflect the stage of oestrus at the time of necropsy and was unrelated to FRET 08-0338 administration.

Macropathology
The macroscopic examination performed at scheduled termination revealed no test substance related lesions. The incidence and distribution of all findings were consistent with the common background seen in Crl:CD(SD) rats at these laboratories.

Histopathology
Changes related to treatment with FRET 08-0338 administration were seen in the kidneys (males) and the liver (females).
Dose descriptor:
NOAEL
Effect level:
274 - 579 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
104 - 197 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: See results

Details on embryotoxic / teratogenic effects:
F1 litter responses
Offspring clinical signs
There were no treatment-related clinical signs seen among the offspring at any dietary inclusion level investigated.

Litter size, sex ratio and survival indices
Among females given 4500 or 15000 ppm, mean implantation counts were slightly, but statistically significantly lower than Control, with 5/10 and 7/10 females having fewer implantation sites than the lowest concurrent Control, and the mean values being outside the HCD range of 15.4 to 17.5 implantation sites. As a consequence, mean litter size in these groups on Day 1 of lactation was lower than Control. The mean number of implantation sites and mean litter size were unaffected at 1500 ppm.
The mean post-partum corpora lutea count recorded on Day 8 of lactation was similar in all groups of females.
Sex ratio, as assessed by the percentage of males present in each litter, and offspring survival from conception to Day 7 of age was unaffected by FRET 08-0338 administration at all dietary inclusion levels investigated.

Offspring body weight
On Day 1 of age, the mean absolute body weight of offspring in litters derived from parent animals given 15000 ppm was 7% and 8% lower than Control for males and females, respectively, with differences attaining statistical significance. The mean body weight gain of these offspring was 30-31% lower than Control throughout Days 1-7 of age, such that mean absolute body weight on Day 7 of age was 20% lower than Control in both sexes.
At 4500 ppm, mean offspring body weights on Day 1 of age were essentially similar to Control. Mean body weight gain between Days 1 and 7 of age was 7-9% lower than Control such that mean absolute body weight on Day 7 of age was 4-5% lower than Control; none of these difference attained statistical significance and no adverse effect of FRET 08-0338 was considered to be inferred.
The body weight and body weight gain of offspring in the 1500 ppm group was considered unaffected by parental treatment with FRET 08-0338.

Offspring macropathology
There were no macroscopic abnormities detected among offspring which died or were killed prior to scheduled termination or at scheduled termination on Day 7 of age that were indicative of an adverse effect of parental treatment with FRET 08-0338.
Abnormalities:
not specified
Developmental effects observed:
not specified

Formulation analysis

Homogeneity was confirmed for FRET 08-0338 in SDS VRF1 certified diet formulations at nominal concentrations of 100 ppm and 15000 ppm:

  • Stability was confirmed at ambient temperature for up to 4 days and frozen for up to 27 days for 100 ppm.
  • Stability was confirmed for up to 22 days at ambient temperature and frozen for 15000 ppm.

The mean concentrations of FRET 08-0338 in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. The percentage difference from the mean values remained within 3% and a coefficient of variation for four samples <14%.

F0 responses

Achieved dose

Mean achieved doses (mg/kg/day) during the study were as follows:

Text Table 1: Achieved doses (mg/kg/day)

 

Males

Females

Dietary Conc. (ppm)

1500

4500

15000

1500

4500

15000

Period

 

 

 

 

 

 

Weeks 1-5 (males)

93

274

941

-

-

-

Females prior to pairing

-

-

-

104

319

957

Females during gestation

-

-

-

116

372

1140

Females during Days 1-7 of lactation

-

-

-

197

579

1805

 

At the highest dietary concentration (15000 ppm) overall average intake was 941 mg/kg/day for the males, 957 mg/kg/day for the females prior to pairing and 1140 mg/kg/day during gestation. During lactation, achieved intake was much higher than in the other study phases, averaging
1805 mg/kg/day, reflecting the increase in female food consumption in response to the increased physiological demands when rearing the litter. Achieved intake in the 1500 and 4500 ppm groups were in proportion to the levels present in the diet.

Histopathology

Changes related to treatment with FRET 08-0338 administration were seen in the kidneys (males) and the liver (females).

Kidney

Slight to moderate hyaline droplets in the cortical tubules was seen in all groups of treated males. Minimal to slight cortical basophilic tubules were seen in males given 4500 or 15000 ppm.

Summary of treatment related findings in the kidney for animals killed after scheduled treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Level (ppm)

0

1500

4500

15000

0

1500

4500

15000

 

 

 

 

 

 

 

 

 

Cortical tubules with hyaline droplets

 

 

 

 

 

 

 

 

Slight

0

5

2

0

0

-

-

0

Moderate

0

1

2

5

0

-

-

0

Total

0

6

4

5

0

-

-

0

 

 

 

 

 

 

 

 

 

 Cortical basophilic tubules

 

 

 

 

 

 

 

 

Minimal

0

0

0

2

0

-

-

0

Slight

0

0

1

1

0

-

-

0

Total

0

0

1

3

0

-

-

0

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

6

5

5

5

0

0

5

 

 

 

 

 

 

 

 

 

 

Liver

Minimal to moderate vacuolation of periportal/diffuse hepatocytes was observed in the liver of females given 4500 or 15000 ppm.

Summary of treatment related findings in the liver after scheduled treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Level (ppm)

0

1500

4500

15000

0

1500

4500

15000

 

 

 

 

 

 

 

 

 

Hepatocyte vacuolation, periportal/diffuse

 

 

 

 

 

 

 

 

Minimal

0

-

-

0

0

0

1

1

Slight

0

-

-

0

0

0

1

1

Moderate

0

-

-

0

0

0

0

1

Total

0

-

-

0

0

0

2

3

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

0

0

5

5

5

5

5

 

 

 

 

 

 

 

 

 

 

Seminiferous tubules were evaluated with respect to their stage in spermatogenic cycle and the integrity of the cell types present within different stages. No cell or stage specific abnormalities were noted.

All other histological changes were considered to be unrelated to treatment.

Conclusions:
Dietary administration of FRET 08-0338 to CD rats for at least 5 weeks in males and approximately 7 weeks in females at concentrations up to and including 15000 ppm was generally well tolerated. Test article related histopathological changes were apparent in the liver of females given 4500 ppm and above, and in the kidneys of males in all treated groups. The liver changes (high values for cholesterol, high values for triglycerides, high adjusted liver weights, elevated alanine aminotransferase activity and hepatocyte vacuolation) observed in females are considered to be related to treatment. These changes may be (partly) due to metabolism of the test substance, and they may also be adverse. The kidney changes detected in the males (hyaline droplets in tubules and cortical basophilic tubules) were consistent with well documented species-specific responses of the male rat in response to the administration with hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. Based on these considerations, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity is based on potentially adverse liver effects and is therefore set at 1500 ppm (equivalent to 93 mg/kg bw/day for males and 104-197 mg/kg bw/day for females).
The reduction in the number of uterine implantations at 4500 and 15000 ppm was minimal (= <15% of Control) and not related to any other changes in reproductive parameters, such as post implantation loss. This effect, however, was statistically significant and outside of the historical control values (representing 10 OECD TG 422 studies). Although this effect may be related to the lower maternal body weight (gain), it is considered to be of uncertain aetiology and therefore potentially adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive performance was 1500 ppm (equivalent to 93 mg/kg bw/day for males and 104-197 mg kg/bw/day for females).
The reduction in birth weight and subsequent body weight gain of the offspring derived from parent animals given 15000 ppm was also of uncertain aetiology, and therefore potentially adverse. It was therefore concluded that the NOAEL for offspring growth, survival and development was 4500 ppm (equivalent to 274 mg/kg bw/day for males and 319 579 mg/kg bw/day for females).
Executive summary:

The developmental toxicity / teratogenicity of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 422 using a screening method. The NOAEL for offspring growth, survival and development was 4500 ppm (equivalent to 274 mg/kg bw/day for males and 319‑579 mg/kg bw/day for females). The NOAEL for reproductive performance was 1500 ppm (equivalent to 93 mg/kg bw/day for males and 104-197 mg kg/bw/day for females).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
104 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information
Justification for selection of Effect on developmental toxicity: via oral route:
The study was conducted on the target substance in vivo, in an appropriate test species and according to internationally recognised guidelines.

Justification for classification or non-classification

Reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females as well as developmental toxicity in the offspring. Reproductive toxicity is subdivided under two main headings, adverse effects on sexual function and fertility, and adverse effects on development of the offspring. Some reproductive toxic effects cannot be clearly assigned to either impairment of sexual function and fertility or developmental toxicity. Nonetheless, substance with these effects, or mixtures containing them, shall be classified as reproductive toxicants.

Hazard categories for reproductive toxicants are divided into two main categories. Category 1 is known or presumed human reproductive toxicants. This category is subdivided into Category 1A, which is a known human reproductive toxicant where the classification is based largely on evidence from humans, and Category 1B, which is a presumed human reproductive toxicant where the classification is based largely on evidence from animal studies. Category 2 is for suspected human reproductive toxicants, where there is some evidence from humans or experimental animals.

Classification is made on the basis of the appropriate criteria and an assessment of the total weight of evidence. Classification is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction and shall not be classified if such an effect is produced solely as a non-specific secondary consequence of other toxic effects. If in some reproductive toxicity studies in experimental animals the only effects recorded are considered to be of low or minimal toxicological significance, classification may not necessarily be the outcome.

 

The test substance was assessed for reproductive and developmental toxicity in a screening study according to OECD Test Guideline 422. The oral administration of the test substance by dietary administration to rats was conducted for a minimum of five weeks, at dietary 1500, 4500 or 15000 ppm (equivalent to a mean achieved dosage of 100, 030 and 1000 mg/kg bw/day respectively).

 

There were no treatment-related clinical signs seen among the offspring at any dietary inclusion level investigated. Among females given 4500 or 15000 ppm, mean implantation counts were slightly, but statistically significantly lower than Control. As a consequence, mean litter size in these groups on Day 1 of lactation was lower than Control. The mean number of implantation sites and mean litter size were unaffected at 1500 ppm. The mean post-partum corpora lutea count recorded on Day 8 of lactation was similar in all groups of females. Sex ratio, as assessed by the percentage of males present in each litter, and offspring survival from conception to Day 7 of age was unaffected by FRET 08-0338 administration at all dietary inclusion levels investigated. The body weight and body weight gain of offspring in the 1500 ppm group was considered unaffected by parental treatment. There were no macroscopic abnormities detected among offspring which died or were killed prior to scheduled termination or at scheduled termination on Day 7 of age that were indicative of an adverse effect of parental treatment with FRET 08-0338. 

The reduction in the number of uterine implantations at 4500 and 15000 ppm was minimal (= <15% of Control) and not related to any other changes in reproductive parameters, such as post implantation loss. This effect, however, was statistically significant and outside of the historical control values. Although this effect may be related to the lower maternal body weight (gain), it is considered to be of uncertain aetiology and therefore potentially adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive performance was 1500 ppm (equivalent to 93 mg/kg bw/day for males and 104-197 mg kg/bw/day for females).

The reduction in birth weight and subsequent body weight gain of the offspring derived from parent animals given 15000 ppm was also of uncertain aetiology, and therefore potentially adverse. It was therefore concluded that the NOAEL for offspring growth, survival and development was 4500 ppm (equivalent to 274 mg/kg bw/day for males and 319‑579 mg/kg bw/day for females).

The effects observed during the study were not conclusively the result of reproductive toxicity of the test item and may be related to other effects. As the aetiology was not certain, the test substance is not classified for reproductive toxicity.