Butanal, 4-(heptyloxy)-3-methyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 22 May 2014 and 4 July 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a relaibility 1 as it has been conducted according to OECD Test Guideline 407 using a dietary study method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA, OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Official notice of J MHLW, METI and ME (21 November 2003), Yakushokuhatsu No. 1121002, Seikyoku No. 2, Kanpokihatsu No. 031121002.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(heptyloxy)-3-methylbutanal

EC Number:

802-100-7

Cas Number:

1093653-57-6

Molecular formula:

C12H24O2

IUPAC Name:

4-(heptyloxy)-3-methylbutanal

Test material form:

other: Liquid

Details on test material:

Test substance: TM 09-217.
Test substance identity (including alternative names): 4-(heptyloxy)-3-methyl butanal.
CAS number: 1093653-57-6.
Intended use: Fragrance ingredient.
Appearance: Liquid.
Storage conditions: At ambient temperature and protected from light.
Supplier: Sponsor

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Identity of treatment groups
The study consisted of one control and three treated groups. Some serial observations needed to be performed without the knowledge of the treatment group; therefore the animal numbering system was such that it was not easy to identify a treatment group from the animal number.

Duration of acclimatisation 15 days before commencement of treatment.
Age of the main study and recovery animals at start of treatment: 50 to 56 days old.
Weight range of the main study and recovery animals at the start of treatment: Males: 240 g to 307 g, Females: 159 g to 228 g

Allocation
Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being
repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Identification of animals
Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.

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 occupants.

Environmental control
Rodent facility
Restricted entry - 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.

Electricity supply
Public supply with automatic stand-by generators.

Animal accommodation
Cages
Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.

Cage distribution
Males and females were blocked by sex and the cages constituting each group were dispersed in the batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The position of the cage batteries in the room were changed weekly, following a rotation plan, to further minimise possible effects of spatial variations.

Number of animals per cage
Five of the same sex (main study and recovery).

Bedding
Wood based bedding which was changed at appropriate intervals each week.

Environmental enrichment
Aspen chew block
Provided to each cage throughout the study and replaced when necessary.
Plastic shelter
Provided to each cage throughout the study and replaced when necessary.

Diet
Rat and Mouse No. 1 Maintenance Diet.
Availability
Non-restricted (removed overnight before blood sampling for haematology or blood chemistry and during the period of urine collection).

Water supply
Supply
Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability
Non-restricted (except during urine collection).

Certificates of analysis
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Method of preparation
The test substance was incorporated into the diet to provide the required concentrations 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.
This premix was diluted with further quantities of plain diet to prepare the two highest concentration test mixes (4900 ppm and 14000 ppm) and a second premix. The second premix was used to prepare the lowest concentration test mix (490 ppm). Each formulation was mixed using a turbula mixer for 200 cycles.

Frequency of preparation
Batches of test diets were prepared each week and divided into daily aliquots.

Storage of preparation
Frozen (nominally -20ºC).

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.

Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 20000 and 10 ppm were analysed to assess the stability and homogeneity of the test substance in the diet matrix.

Achieved concentration
Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analysed for achieved concentration of the test
substance.

Administration
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:

Experimental Procedure
Analytical procedure
Apparatus and instrumentation
Gas chromatograph (GC): Shimadzu GC-2010 Plus Gas Chromatograph with a Shimadzu AOC-20i Autosampler and FID detector
Chromatography data handling: Waters Empower 2, Build 2154, Feature Release 4, Service Pack D
Sample registry system: Huntingdon Life Sciences, Version 1.0.8.0
Test substance management: Pristima, Xybion Medical Systems
Corporation, Version 6.3.2 and 6.4.0
Balances fitted with printers: Capability of weighing to 2, 5 or 6 decimal places
General laboratory apparatus and glassware.
Reagents
Control vehicle: Rat & Mouse No1 Maintenance diet
Acetone: Glass distilled grade
Internal Standard: Dimethyl phthalate dissolved in acetone at 500 μg/mL

Preparation of standards
A primary standard solution (250 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 25 mg) of TM 09-217 in acetone (100 mL). A secondary standard (25 μg/mL) was prepared from the primary by 20-fold dilution in acetone. Solutions for instrument calibration were prepared by appropriate dilution of the secondary standard using acetone and contained TM 09-217 at nominal concentrations of 0.5 μg/mL, 1 μg/mL, 1.5 μg/mL, 2 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL and 12.5 μg/mL. To an accurate 1 mL volume of each calibration standard, 100 μL of internal standard solution was added prior to injection onto the GC.

Sample process
A representative sample of diet formulation (nominally 10 g) was accurately weighed and extracted (mechanical shake, 30 minutes) in an accurate volume of acetone (100 mL). The extract was centrifuged (3500 rpm, 5 minutes) and then diluted using acetone as appropriate, to provide a solution containing TM 09-217 at an expected concentration of 1 μg/mL. To an accurate 1 mL volume of each sample, 100 μL of internal standard solution was added prior to injection onto the GC.
The concentration of TM 09-217 in the final solution was quantified by GC using FID detection as detailed in the chromatographic section.

Procedural recoveries
At each analytical occasion recoveries were determined 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 TM 09-217. The prepared procedural recoveries were analysed in accordance with the analytical procedure. Procedural recoveries were used to correct analytical values.
The concentration of TM 09-217 in the final solution was quantified by gas chromatography using FID detection as detailed in the following section.
Typical chromatographic conditions
Column: HP-1, 60 m × 0.25 mm id. Film thickness 0.25 μm
Injector temperature: 300 ºC
Column temperature: Initial: 80ºC (hold for 1 min)
Ramp 1: 10ºC / minute to 210ºC
Ramp 2: 30ºC / minute
Final 1: 300ºC (hold for 2 min)
Detector temperature: 300 ºC
Hydrogen: 40 mL/min
Air: 400 mL/min
Make up: Helium: 30 mL/min (constant flow)
Injection mode: Splitless
Injection volume: 1 μL (2 sample washes, 3 sample pumps)
Detector type: Flame Ionisation
Carrier gas: Helium: 1.49 mL/min (constant flow)
Injector purge: Helium: 3 mL/min
Run time: 19 minutes
Calculation
The peak area for the TM 09-217 peak was calculated, and the peak area ratio response to the internal standard peak area was measured. Calibration curves (with 1/x weighting) were constructed by linear regression of standard response versus standard concentration. The area ratio responses of the peaks observed at the characteristic retention times for TM 09-217 and the internal standard in sample and procedural recovery chromatograms was measured. The concentration of TM 09-217 was determined using the following equation:
Analysed concentration, ppm = (Y-I / S) x (V/W)
Procedural recovery values were calculated using the following equation:
Procedural recovery value % (analysed concentration, ppm / fortified concentration ppm) x 100
Sample concentrations were corrected for the mean procedural recovery value at analysis using the following equation:
Corrected concentration, ppm = Analysed concentration, ppm  100
Where
Y = Peak area response ratio for TM 09-217 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 = Weight of sample (g)
R = Mean procedural recovery percentage 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 quantification, 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 10 ppm and 20000 ppm.

Homogeneity and stability in RM1 diet formulations
Prior to treatment, the homogeneity and stability of TM 09-217 in RM1 diet formulations was assessed at the potential lowest and highest concentrations intended for feeding.
Specimen batches of RM1 diet formulation, containing TM 09-217 at nominal concentrations of 10 ppm and 20000 ppm were prepared by Pharmacy personnel and randomly sampled in duplicate from the top, middle and bottom of the turbula drum. Five extra samples were taken from the drum for stability determination.
Homogeneity: The duplicate samples taken from the top, middle and bottom of the treated diets were analysed singly in accordance with the analytical procedure. The mean analysed concentration was taken as the initial stability timepoint.
Stability: The five extra diets taken for stability determination were stored at ambient temperature (nominally 21°C) for 1, 2, 4 and 8 days and for 22 days frozen (nominally -20°C) following preparation. The diet stability samples taken from storage were frozen (nominally -20°C) pending analysis.
The Day 1 and Day 8 samples were analysed initially and following a review of this data where only 24 hours ambient stability was achieved, the Day 2 and Day 4 samples were analysed as well as the scheduled Day 22 samples. Each sample was analysed in duplicate in accordance with the analytical procedure.
Concentration in test formulations
At Weeks 1 and 4 of treatment, representative samples (200 g) of test diet 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.

Results
Method validation
The analytical procedure was successfully validated for TM 09-217 in RM1 diet with respect to the specificity of chromatographic analysis, limit of detection, limit of quantification, 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 TM 09-217 in the control sample chromatogram;
The limit of detection was estimated as 0.0189 ppm using the operating parameters defined in this procedure;
The limit of quantification was estimated as 0.0632 ppm using the operating parameters defined in this procedure;
Linearity was confirmed over the nominal concentration range 0.5 μg/mL to 12.5 μg/mL with a coefficient of determination >0.999;
The precision of injection was <1% for six replicate injections of standard solutions containing TM 09-217 at nominal concentrations of 0.5 μg/mL and 12.5 μg/mL;
Method accuracy and precision were confirmed and results are detailed:
a mean procedural recovery value of 101.9% (CV=2.13%, n=6) was obtained for
10 ppm and 96.4% (CV=1.24%, n=6) was obtained for 20000 ppm.

Formulation trial
The homogeneity of TM 09-217 in RM1 diet formulations was confirmed at nominal concentrations of 10 ppm and 20000 ppm. Each formulation achieved an accuracy within 14% of the nominal concentration and a precision, measured by the coefficient of variation, of <3%.
The stability of TM 09-217 in RM1 diet formulations at nominal concentrations of 10 ppm and 20000 ppm was confirmed for 2 days at ambient temperature storage followed by 9 days frozen storage. Stability was also confirmed for up to 22 days frozen storage The mean analysed concentration remained within 9% of the initial time zero value.

Concentration in dose formulations
The mean concentrations were between -1.8% and +9.3% of nominal values which is within applied limits of +10%/-15%, confirming the accuracy of formulation.
Precision of individual results from mean values is <5% with two exceptions (Week 1, groups 2 and 4 at 5.09% and 5.55% respectively). Three of the four the 4 individual results for these groups are within applied limits in terms of concentration. Procedural recovery values remained within 96.5% and 103.4%, confirming the continued accuracy of the method.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, linearity of detector response, precision of injection, accuracy and precision. Homogeneity was confirmed for TM 09-217 in RM1 diet formulations at nominal concentrations of 10 ppm and 20000 ppm. Stability was confirmed at ambient storage for 2 days following 9 days frozen storage and for up to 22 days frozen storage. The mean concentrations of TM 09-217 in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Minimum period 28 days of treatment followed by a 14 day recovery period. The last dose was administered on Day 28 for males and on Day 29 for females.
The surviving main study animals were killed on Day 29 or Day 30. The recovery phase commenced on Day 29 for males and Day 30 for females; these animals were killed on Day 43 for males and Day 44 for females (Day 15 of recovery). Serial observations were recorded at appropriate intervals.

Frequency of treatment:

Continuously. During the recovery period, all animals were given untreated diet.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 490, 4900 and 14000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

5/sex/dose with an additional 5/sex/dose for the two recovery groups.

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

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

Detailed physical examination and arena observations
Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behaviour 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 all animals during Week 4 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing. The following measurements, reflexes and responses were recorded:
Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 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 force indicators. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 4 of treatment the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.5).
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Body weight
The weight of each animal was recorded one week before treatment commenced (Week P1), on the day that treatment commenced (Week 0), weekly throughout the study (the last weight recorded during the treatment period was on Day 28 for males and on Day 29 for females) and before necropsy.

Food consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1) and for each week throughout the study (daily during the treatment period and weekly during the recovery period). Food consumption was not recorded for the period Day 26 to 27 of treatment in error for Cage 1 (Group 1 male).

Water consumption
Fluid intake was assessed by daily visual observation. On five occasions during the course of the study, Group 4M consumed more water than Group 1M, however, the effect was intermittent and only in one sex. Consequently quantitative measurements were not performed.

Oestrous cycles - vaginal smears
Dry smears Daily smears were taken for 15 days during the treatment period (during Weeks 3 and 4 of treatment) and for eight days during recovery (Week 2 of recovery), using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the oestrous cycle.

Haematology, peripheral blood
Blood samples were collected after overnight withdrawal of food at the following occasions:
At termination: All main study animals
Day R15 All recovery phase animals.

Blood sampling was performed on the morning after overnight collection of urine. Animals, were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Animals were held under light general 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
Macrocytosis
Microcytosis
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:
At termination: All main study animals
Day R15 All recovery phase animals.

Blood sampling was performed on the morning after overnight collection of urine. Animals, were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Animals were held under light general 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)
Gamma-glutamyl transferase (gGT)
Total bilirubin (Bili)
Total 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)

The following electrophoretic protein fractions were processed using a Helena SPIFE 3000 with agarose gel and scanning with a suitable densitometer:
Albumin (Alb)
1 globulin (a1)
2 globulin (a2)
 globulin (Beta)
 globulin (Gamma)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.
An additional 0.5 mL of whole blood was collected and placed in tubes containing EDTA. The plasma was separated by centrifugation (1500 g, five minutes, +4C) to provide a minimum of 0.2 mL of plasma. The plasma samples were stored frozen (-20C) pending any requirement for analysis for T3, T4 and TSH. In the absence of any histopathological changes in the thyroid, no analysis was required of these samples and they will be discarded
after finalisation of this report.

Urinalysis
Animals were placed in an individual metabolism cage overnight, without food or water. Urine samples were collected over approximately 16 hours at the following occasions:
At termination: All main study animals
Day R15 All recovery phase animals.

The individual samples were examined for the following characteristics:

Using manual methods:
Clarity and Colour (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips, interpreted using a Clinitek®500 instrument:
Ketones (Keto)
Bilirubin/bile pigments (Bili)
Blood pigments (UBld)
Urobilinogen (Urob)
Using a Roche P Modular analyser:
Protein (T-Prot)
Creatinine (T-Creat)
Glucose (T-Gluc)
Sodium (T-Na)
Potassium (T-K)
Chloride (T-Cl)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
Epithelial cells (Epi)
Leucocytes (WBC)
Erythrocytes (RBC)
Crystals (Cryst)
Casts
Spermatozoa (Sperm)
Other abnormal components (A)

Sacrifice and pathology:

Terminal Procedures
All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.
Schedule ; Main study animals were killed following four weeks of treatment (Day 29 for males and Day 30 for females). Recovery animals were killed following four weeks of treatment and two weeks of recovery (Day 15 of recovery).
Method of kill; Carbon dioxide asphyxiation with subsequent exsanguination.
Sequence; To allow satisfactory inter-group comparison.

Organ weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals.
Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of
those detailed below:
Right testes and right epididymis; In modified Davidson’s fluid.
Eyes; In Davidson’s fluid.

Histology
Processing; Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List; Main study animals of Groups 1 and 4 killed at a scheduled interval.
Abnormalities only; All animals.
Liver and kidneys only; All main study females of Group 2 and 3 and all recovery females of Group 1 and 4.
Routine staining; Sections were stained with haematoxylin and eosin.

Light microscopy
Tissues preserved for examination were examined as follows:
Scheduled kill:
Main study
Allanimals of groups 1 and 4. - All specified tissues.
All animals of groups 2 and 3 - Abnormalities only.

Recovery
An animals of Groups 1 and 4 - Abnormalities

The following tissues, which were considered to exhibit a reaction to treatment at the high dose, were examined for all main study and recovery females:
Liver and kidneys
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.

Sperm analysis
Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
The following tests were performed:

Sperm motility – all groups
A sample of sperm was expressed from the vas deferens into pre-warmed (37oC) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 μm depth cannula by capillary action and at least 200 sperm per animal analysed using
the Hamilton Thorne IVOS Computer Assisted Sperm Analyser (CASA).

Sperm morphology – all groups
A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male.

Sperm count – all groups
The left cauda epididymis of each male was weighed; those of Groups 2 and 3 were frozen. Prior to analysis the cauda epididymis was allowed to thaw
if necessary. The left cauda epididymis of each male was then homogenised for at least one minute in 10 mL of a mixture of 0.9% saline, 0.01% merthiolate and 0.05% Triton X-100 (SMT). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count
using CASA.

Homogenisationresistant; spermatid count – all groups
The left testis of each male of Groups 2 and 3 was frozen. Prior to analysis the testis was allowed to thaw if necessary. The left testis of each male was homogenised for at least two minutes in 25 mL of SMT. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA.

Other examinations:

Serial observations
Proof of absorption
Blood samples were obtained from animals. The sampling schedule was as follows:
Week 4: All main study animals

Time of sampling Approximately one hour after the 12 hour dark cycle had finished.
Blood sample site: Tail vein.
Anaesthetic: Isoflurane.
Anticoagulant: Lithium heparin.
Blood volume: 0.5 mL.
Treatment of samples: Stored on wet ice prior to centrifugation.
Centrifugation conditions: At 2000g for ten minutes at 4°C.
Number of aliquots per sample: One.
Temporary storage conditions: Placed on dry ice prior to transfer to freezer external to animal facility.
Final storage conditions: Deep frozen (approximately -70ºC), protected from light.
Fate of plasma samples: Despatched to the sponsor
Bioanalysis: Performed by the sponsor, the results from the analysis performed by the sponsor were not reported as part of this study, but may be reported in a separate study by the sponsor.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See Results

Mortality:

mortality observed, treatment-related

Description (incidence):

See Results

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Results

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See Results

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See Results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Results

Histopathological findings: neoplastic:

no effects observed

Details on results:

Detailed physical examination and arena observations
The appearance and behaviour of the animals were unaffected and there were no deaths during treatment.

Sensory reactivity and grip strength
The sensory reactivity and grip strength investigations did not identify any findings that could be ascribed unequivocally to treatment with TM 09-217. The group mean forelimb and hindlimb grip strength for males and forelimb grip strength for females receiving 14000 ppm were slightly low compared with those of controls but statistical significance (p<0.05) was not attained, therefore this finding was considered incidental to treatment.

Motor activity
Motor activity assessment in Week 4 of treatment revealed slightly reduced overall high beam (rearing) and low bean (cage floor activity) scores in all treated groups. There were however, no statistical significance attained and no dose-relationship apparent, therefore this was considered incidental to treatment.

Body weight
Body weight gains were reduced, when compared with control values, during the first two weeks of treatment in females receiving 14000 ppm (61% of Control for both weeks), with the overall reduction in weight gain (Week 1 to 4) being approximately 78% of Control. Females in the lower dose groups also showed some body weight gain variation during treatment; however, the overall body weight gains were similar to controls. Additionally, body weight gains were slightly reduced, when compared with control values, during the first week of treatment for males receiving 14000 ppm (81% of Control). During Week 2 to 4 of treatment, however, the bodyweight gains were similar to controls and the overall body weight gain was only slightly reduced (87%). During the recovery period, the bodyweight gains for males and females which had previously received 14000 ppm were similar or above control values. There was no effect on bodyweight for males given 490 or 4900 ppm.

Food consumption
There was a very slight reduction in food consumption, compared with control and pretreatment values, for males receiving 14000 ppm during the majority of the treatment period. During recovery, however, food consumption for males previously given 14000 ppm was similar to controls.
There was no treatment-related effect on food intake for treated females and males receiving 490 or 4900 ppm.

Oestrous cycles
There was no effect of treatment for oestrous cycles on treated females.

Haematology
There were no treatment-related changes seen in Week 4 haematology investigations. Changes that were seen, but were not considered to be related to treatment included mean total leucocyte counts were increased in males receiving 14000 ppm, with statistical significance obtained. Associated with this, there was an increase in neutrophil, lymphocyte, eosinophil, basophil and monocyte counts, in males given 14000 ppm. After 2 weeks of recovery, assessment of the haematological investigations revealed values that were similar to controls in males previously receiving 14000 ppm, which indicates recovery in the findings seen after 4 Weeks of treatment.
All other inter-group differences from controls, including those which attained statistical significance, were generally minor, lacked dose-relationship or were confined to one sex. Such differences include, but were not limited to, the slightly increased haemoglobin concentration in males receiving 14000 ppm, but in females at this dose level haemoglobin concentration was slightly lower than the control value. In addition mean cell haemoglobin concentration, prothrombin time and activated partial thromboplastin time were decreased in females receiving 14000 ppm, but there was no apparent dose-relationship and no similar findings in the males.

Blood chemistry
The biochemical examination of the blood plasma at the end of a four week treatment period revealed, when compared with the controls, reduced triglycerides concentrations in males receiving 14000 ppm. There was, however, no similar findings seen in females. After 2 weeks of recovery, males previously given 14000 ppm revealed triglyceride values similar to Controls, which indicates recovery in this finding. Changes in plasma electrolyte levels that were attributed to treatment included statistically significantly increased sodium, calcium and phosphorus levels in treated males. A review of the individual data indicated that the majority of individual values were above the highest individual control value; however there was no dose-relationship apparent. In addition treated females indicated an increase in potassium levels, with statistical significance achieved for all treated group, there was, however no dose-relationship apparent. After 2 weeks of recovery, males and females previously given 14000 ppm, revealed plasma electrolyte values similar to controls, which indicate recovery in these findings.
Albumin/globulin ratio was slightly decreased in treated males, with a dose relationship apparent and statistical significance achieved for males given 14000 ppm. Contrary to this, however, females receiving 14000 ppm revealed slightly high albumin/globulin ratio. After 2 weeks of recovery, albumin/globulin ratio were still slightly reduced for males previously receiving 14000 ppm, in contrast, females previously given 14000 ppm revealed albumin/globulin ratio similar to the controls.
All other inter-group differences from controls were minor, lacked dose relationship, were inconsistent between the sexes or were considered of no toxicological significance and were therefore attributed to normal biological variation. Such differences included the increased alkaline phosphatase, alanine amino-transferase and aspartate amino-transferase levels in males receiving 14000 ppm, as the majority of individual values were within the background control range (90-percentile range 111 to 259 U/L for alkaline phosphatase, 30 to 74 U/L for alanine amino-transferase, and 57 to 96 U/L for aspartate amino-transferase; n = 325) and no similar finding was observed in the females. In addition, the statistically significant decreased bile acid concentrations in treated females, as the majority of individual values were within the background control range (90-percentile range 7.1 to 67.0 µmol/L; n = 35), and there was no dose-relationship apparent.

Urinalysis
Examination of the urine after 4 weeks of treatment revealed a decrease in urinary volume and pH, when compared with the controls, in males receiving 14000 ppm. Urinary volume was also low for males receiving 490 or 4900 ppm and pH was also decreased for females given 14000 ppm.
Specific gravity was increased when compared with the control values, in males given 14000 ppm. In contrast to this, total creatinine, chloride, sodium and potassium were low when compared to the control values for all treated male groups, with the majority of individual values below the concurrent control range, although there was no dose-relationship.
In addition, ketones were present in the urine of majority of treated males, generally at small amounts in all groups. Accompanying these findings was change in the appearance of the urine samples, since the samples from the control animals appeared pale yellow, whilst in some treated males the urine appeared medium yellow, or cloudy pale yellow.
After 2 weeks of recovery, assessment of the urine revealed values that were similar to controls in males previously receiving 14000 ppm.
All other inter-group differences from controls were minor, lacked dose relationship, were inconsistent between the sexes or were considered of no toxicological significance and were therefore attributed to normal biological variation.

Sperm analysis
Examination of sperm parameters revealed changes at 14000 ppm.
Sperm and spermatid numbers at 14000 ppm were lower than those of the Control group; these changes were statistically significantly for the spermatid numbers only. This would suggest an adverse effect on sperm production. Statistical significance was achieved only for the beat cross frequency (BCF) value. Two weeks after cessation of treatment with TM 09-217 there was evidence of recovery. Beat cross frequency however, remained statistically significantly lower than that of the Control group.
Sperm motility, morphology and concentration were unaffected by treatment with TM 09-217 at dietary levels up to 4900 ppm. Changes in percentage motile and progressively motile sperm, percentage normal morphology and most sperm motion parameters were observed after treatment with TM 09-217 at a dietary level of 14000 ppm. These changes were however, largely due to one animal (No. 11) with immotile sperm. Therefore, it was
considered, that this was not treatment related.

Organ weights
The analysis of organ weights after 4 weeks of treatment revealed, when compared with the controls, increased bodyweight adjusted kidney and liver weights in males and females receiving 14000 ppm, with statistical significance attained for liver weights in both sexes and for kidney weights in females. Females receiving 4900 ppm, also revealed statistically significant increased bodyweight adjusted kidney weight. At the end of the two week
recovery period, absolute and adjusted kidney weights were high in males and females previously given 14000 ppm, with statistical significance being attained in both sexes. In addition, after 2 weeks of recovery, absolute and adjusted liver weights remained high in males previously receiving 14000 ppm, but in females at this dose level, liver weights were similar to controls.
There was also an increase in bodyweight adjusted spleen and thymus weights for males receiving 14000 ppm; however, statistical significance was only obtained for the thymus weights. After 2 weeks of recovery, bodyweight adjusted spleen and thymus weights were similar to controls in males previously receiving 14000 ppm.
There were no other differences from controls that were attributed to the administration of TM 09-217. There was no effect of treatment for males receiving up to 4900 ppm.

Macropathology
Animals killed after 4 weeks of treatment The macroscopic examination performed after 4 weeks of treatment revealed no test
substance related lesions.
The incidence and distribution of all findings were consistent with the common background seen at these laboratories.

Animals killed after 2 weeks of recovery
The macroscopic examination performed after 2 weeks of recovery revealed no test substance related lesions.
A mass was identified in the left epididymis of animal No. 25 previously given 14000 ppm. This epididymis was not examined histologically as it was used for sperm analysis.
All findings were considered to be incidental and unrelated to treatment.

Histopathology
Animals killed after 4 weeks of treatment
Treatment related findings
Changes related to treatment with TM 09-217 were seen in the livers and kidneys of female animals treated for 4 weeks.
Liver
Minimal to slight centrilobular hypertrophy was seen only in the livers of female animals given 14000 ppm.

Kidney
An increased incidence and severity of tubular basophilia was seen in the kidneys of female animals given 4900 or 14000 ppm when compared with controls.

Incidental findings
A slightly increased incidence and severity of plasmacytosis was observed in the mandibular lymph node of male animals given 14000 ppm when compared with controls. This microscopic finding, however, is a very common background change and often correlates to the enlarged mandibular lymph nodes observed at necropsy. In this study there were no histopathological findings involving other lymphatic tissues, so this incidence pattern is
considered fortuitous rather than treatment-related. The incidence and distribution of all other findings were consistent with the common
background of microscopic changes seen at these laboratories.

Animals killed after 2 weeks of recovery
Complete recovery from the centrilobular hypertrophy observed in the livers of females given 14000 ppm occurred after 2 weeks respite from treatment. Tubular basophilia was still present in the kidneys of females given 14000 ppm after 2 weeks respite from treatment. However, a partial recovery from the change was observed as the severity was only minimal.

Kidney
Minimal tubular basophilia was seen in the majority of female animals given 14000 ppm after the 2 week recovery period.

Discussion
The dietary administration of TM 09-217, a fragrance ingredient, to Crl:CD (SD) rats for 4 weeks at dietary concentrations up to 14000 ppm was generally well tolerated and the main target organs were identified as the liver, kidneys and testis (sperm production). There were no deaths or adverse clinical signs at any dietary level, but during the first weeks of treatment body weight gains were reduced in animals receiving 14000 ppm, and food
consumption was reduced in males given 14000 ppm. This was attributed to a non-specific toxic response at this level as body weights and food intake were similar to that of the Controls for the remainder of the treatment period.
An effect of treatment upon the liver was indicated by the increased liver weight reported for males and females receiving 14000 ppm, and the associated centrilobular hypertrophy revealed at microscopic examination in females at this dose level. No similar histopathological change was observed in the treated males, however, very minimal generalized hypertrophy may sometimes be present but not always be easy to detect microscopically. Some findings occurred in the study that may be consequences of the effect upon liver function. Such findings included the reduced plasma triglyceride concentration in males given 14000 ppm, and the trend towards the increased plasma electrolyte levels (sodium, calcium, phosphorus and potassium) in treated males or females given 14000 ppm.
In addition, the decreased albumin/globulin ratio in males receiving 14000 ppm, or increased albumin/globulin ratio in females at this dose level, may also be a consequence of an effect upon the liver. A complete recovery from the centrilobular hypertrophy observed in the females occurred after 2 weeks respite from treatment and therefore the finding was not considered adverse.
The kidney was also identified as a target organ since the macroscopic examination after four weeks of treatment revealed an increased incidence and severity of tubular basophilia in the kidney of females given 4900 or 14000 ppm when compared with controls. In both sexes, kidney weights were increased at 14000 ppm, and in females receiving 4900 ppm. There were, however, no detectable histopathological findings in the kidney of both sexes to correlate with the increased body weight adjusted kidney weights reported at necropsy. The change was still present in the kidneys of females given 14000 ppm after 2 weeks respite from treatment, however, the tubular basophilia observed after the recovery period was only minimal. Some findings were revealed during the examination of urine after four weeks of treatment which may be a consequence of an effect upon the kidneys. Such findings included a decrease in urinary volume and pH, increased specific gravity and urinary proteins in males receiving 14000 ppm and in contrast, low creatinine, chloride, sodium and potassium for treated male groups. In addition, ketones were present in the urine of the majority of the treated males and there was a change in the appearance of the urine samples, which may also be a consequence of an effect upon the kidney. None of these findings, were, however, considered adverse.
Sperm and spermatid numbers at 14000 ppm were statistically significantly lower than those of the Control group, which would suggest an effect on sperm production and therefore, were considered adverse. Statistical significance was achieved only for the BCF value, which did not show recovery after cessation of treatment. Microscopic examination revealed no detectable changes in the testes or epididymides to correlate with these findings obtained from treated males. In addition there were no histopathological changes observed in the reproductive tract tissues of male or female animals given 14000 ppm for 4 weeks. As there were no corresponding changes to the lower sperm and spermatid numbers, this would suggest that these changes were sperm specific. A further study would need to be conducted to investigate the significance of these findings.
The cause of the increase in spleen and thymus weights for males receiving 14000 ppm was not established.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

368 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on the findings in the sperm.

2

Dose descriptor:

NOAEL

Effect level:

1 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Liver and kidney findings were considered non-adverse.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Formulation analysis

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, linearity of detector response, precision of injection, accuracy and precision. Homogeneity was confirmed for TM 09-217 in RM1 diet formulations at nominal concentrations of 10 ppm and 20000 ppm. Stability was confirmed at ambient storage for 2 days following 9 days frozen storage and for up to 22 days frozen storage. The mean concentrations of TM 09-217 in test formulations analysed for the study was within +10%/-15% of nominal concentrations, confirming accurate formulation.

Achieved dose

The overall achieved dosages during treatment were 38, 368 and 1061 mg/kg/day for males given 490, 4900 and 14000 ppm, respectively, and 43, 397 and 1150 mg/kg/day for females receiving 490, 4900 and 14000 ppm, respectively.

Summary of treatment related findings in the liver for animals killed after 4 weeks of treatment

Group / sex	1 M	2 M	2 3	4 M	1 F	2 F	3 F	4 F
Level (ppm)	0	490	4900	14000	0	490	4900	14000
Hypertrophy, centrilobular:
Minimal	0	-	-	0	0	0	0	3
Slight	0	-	-	0	0	0	0	1
Total	0	-	-	0	0	0	0	4
Number of tissues examined	5	0	0	5	5	5	5	5

 

Summary of treatment related findings in the kidney for animals killed after 4 weeks of treatment

Group / sex	1 M	2 M	2 3	4 M	1 F	2 F	3 F	4 F
Level (ppm)	0	490	4900	14000	0	490	4900	14000
Basophilia, Tubular
Minimal	1	1	-	2	1	1	4	3
Slight	0	0	-	0	0	0	1	1
Total	1	1	-	2	1	1	5	4
Number of tissues examined	5	1	0	5	5	5	5	5

 

Summary of treatment related findings in the kidney for animals killed after 2 weeks of recovery

Group / sex	1F	4F
Level (ppm)	0	14000
Basophilia, Tubular
Minimal	1	4
Total	1	4
Number of tissues examined	5	5

Applicant's summary and conclusion

Conclusions:

It is concluded that dietary administration of TM 09-217 to Sprague-Dawley rats at dietary
concentrations of 490, 4900 or 14000 ppm (equivalent to 38, 368 and 1061 mg/kg/day for
males and 43, 397 and 1150 mg/kg/day for females, respectively) for four weeks was
generally well-tolerated. There was an initial reduction in body weight gain and food intake,
particularly during the first week. The liver and kidney were identified as target organs, and
reduced sperm production was evident in males given 14000 ppm. Based on the findings in
the sperm the NOAEL for the males in this study was considered to be 4900 ppm
(368 mg/kg/day). For the females, as the liver and kidney findings were considered
non-adverse, the NOAEL was considered to be 14000 ppm (1150 mg/kg/day).

Executive summary:

The repeated dose toxicity for the test substance, TM 09-217, was assessed according to OECD Test Guideline 407. Three groups, each comprising five male and five female Crl:CD(SD) rats, received TM 09-217 orally, via the diet, at concentrations of 490, 4900 or 14000 ppm for four weeks. A similarly constituted control group received the vehicle, untreated diet, throughout the treatment period. A further five male and five female rats were assigned to each of the Control and high dose groups. These animals were treated for four weeks, followed by a two week period without treatment to assess the potential for any treatment-related change to recover. During the study, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (visual assessment only), oestrous cycles, haematology, blood chemistry, urinalysis, sperm analysis, organ weight, macropathology and histopathology investigations were undertaken. There was an initial reduction in body weight gain and food intake, particularly during the first week. The liver and kidney were identified as target organs, with increased bw adjusted kidney and liver weight at the high dose (male and female) and middle dose (female). The microscopic changes included minimal to slight centrilobular hypertrophy in the liver (14000 ppm) and increased incidence and severity of tubular basophilia in the kidney (4900 and 14000 ppm), all were seen in the females. Sperm and spermatid numbers at 14000 ppm were lower than those of the control group. Based on the findings in the sperm the NOAEL for the males in this study was considered to be 4900 ppm (368 mg/kg/day). For the females, as the liver and kidney findings were considered non-adverse, the NOAEL was considered to be 14000 ppm (1150 mg/kg/day).