4,7-Methano-1H-indene-5-acetaldehyde, octahydro-

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 30 June 2014 and 02 March 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 407 using a continuous dietary admixture 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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[(8R)-tricyclo[5.2.1.0²,⁶]decan-8-yl]acetaldehyde; 2-[(8S)-tricyclo[5.2.1.0²,⁶]decan-8-yl]acetaldehyde

EC Number:

940-300-7

Cas Number:

1339119-15-1

Molecular formula:

C12H18O

IUPAC Name:

2-[(8R)-tricyclo[5.2.1.0²,⁶]decan-8-yl]acetaldehyde; 2-[(8S)-tricyclo[5.2.1.0²,⁶]decan-8-yl]acetaldehyde

Test material form:

other: Liquid

Details on test material:

Identification : IFF TM 11-213
Physical State/Appearance : Clear colorless liquid
Date Received : 19 June 2014
Storage Conditions : Approximately 4 °C in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for five days during which time their health status was assessed. A total of sixty animals (thirty males and thirty females) were accepted into the study. At the start of treatment the males weighed 185 to 219g, the females weighed 138 to 158g, and were approximately six to eight weeks old.

The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A powdered diet (Rat and Mouse SQC Ground Diet No. 1, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from
Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

For the purpose of this study the test item was incorporated into the diet at concentrations of 0,
300, 600, and 1200 ppm as follows:
A known amount of test item was mixed with a small amount of basal laboratory diet until
homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to
a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant
speed, setting 1 in a Hobart QE 200 mixer.

The stability and uniformity of distribution of the test item in the diet were determined by Harlan
Laboratories Ltd., Shardlow, UK, Analytical Services. Results showed the dietary admixtures to
be stable for 12 days at 300 ppm and 21 days at 1200 ppm. Dietary admixtures were prepared
prior to the first treatment, and weekly thereafter. The diet was stored in labelled, double plastic
bags in labelled, covered plastic bins at approximately -18 °C and fed daily. Samples were taken
from the dietary admixtures and analysed for uniformity of distribution and concentration at
Harlan Laboratories Ltd, Shardlow, UK, Analytical Services. The results indicate that the
mean prepared dietary admixture concentrations were within ± 2% of the nominal concentration.

The test item was administered continuously, for twenty-eight consecutive days, by dietary
admixture. Control animals were treated in an identical manner with basal laboratory diet.
Recovery group animals were maintained for a further fourteen days treatment-free period
following termination of treatment.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Introduction
The test item concentration in the test sample was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of two peaks.

Test item
The test item described in the main part of this study was also used as the analytical standard.

Analytical procedure
Preparation of standard solutions
Stock solutions of test item in acetonitrile were prepared for external standard calibration. An aliquot, 100 mg of test item, was accurately weighed into a 100 mL volumetric flask and brought to volume with acetonitrile to yield a solution with a concentration of 1000 ppm. Aliquots of this stock standard solution were used to prepare working standard solutions in acetonitrile with a concentration of 100 ppm.
On each occasion, standard solutions derived from two stock standard solutions were used for calculations.

Analysis of samples
The dietary admixtures received were extracted with acetonitrile. An aliquot, 5 g of dietary admixture was accurately weighed into a suitable jar and the correct amount of acetonitrile added. This was then ultrasonicated for 15 minutes and left to extract overnight in ambient conditions. The sample solutions were then ultrasonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with acetonitrile to achieve the working concentration.

Preparation of accuracy samples
Samples of diet were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared to analysis as for the test samples.

Preparation of linearity standards
A range of standard solutions were prepared in acetonitrile from a stock solution of 1083 ppm by serial dilution covering the concentration range of 0 to 163.45 ppm.

Instrument set up
GC system: Agilent Technologies 5890, incorporating autosampler and work station.
Column: ZB-5 (30 m x 0.53 mm id x 5 µm film).
Oven temperature program: Oven: 130 °C for 0 minutes with 10 °C/minute to 250 °C for 0 minutes, then 500 °C/minute to 300 °C for 5 minutes.
Injection temperature: 250 °C
Flam ionisation detector temperature: 250 °C
Injection volume: 1 µL
Retention time: ~8 and 9 mins.

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical method
Specificity
The control dose samples and an analyzed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. The data was found to have a linear correlation with the calibration range. The R2 fit of the calibration curve to the data was 0.999 and considered to be acceptable.

Accuracy
The fortified samples of diet were found to have a recovery value of ± 10 % of the fortification.
Test item dietary admixtures
The admixtures investigated during the study were found to comprise test item in the range of 98 % to 102 % and thus the required content limit of ± 10 % with reference to the nominal content was met.
The test item was found to be stable in the admixtures when kept frozen (300 ppm 12 days, 1000 and 20000 ppm 21 days) due to results which met the variation limit of 10 % from the time-zero mean.
In conclusion, the results indicate the accurate use of the test item and diet as vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was proven.

Discussion
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.

Duration of treatment / exposure:

Twenty-eight consecutive days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 300, 600, 1200 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

5 / sex / opse

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change daily from
the start of treatment. All observations were recorded.

Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 27, all animals were observed for signs
of functional/behavioral toxicity. Functional performance tests were also performed on all
animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built
arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The
scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral
Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used
to assess motor activity. Animals of one sex were tested at each occasion and were randomly
allocated to the activity monitors. The tests were performed at approximately the same time
each occasion, under similar laboratory conditions. The evaluation period was one hour for each
animal. The time in seconds each animal was active and mobile was recorded for the overall one
hour period and also during the final 20% of the period (considered to be the asymptotic period,
Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength.
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was
pulled by the base of the tail until its grip was broken. The animal was drawn along the trough
of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force
required to break the grip for each animal was made. Three consecutive trials were performed
for each animal. The assessment was developed from the method employed by Meyer et al
(1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and
proprioceptive stimuli. This assessment was developed from the methods employed by Irwin
(1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring
System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

Body Weight
Individual body weights were recorded on Day 1 (prior to the start of treatment) and at weekly
intervals thereafter. Body weights were also performed prior to terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.
Food conversion efficiency and mean achieved dosages were calculated retrospectively.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles
for any overt changes except during Week 3 where water intake was measured gravimetrically.

Laboratory Investigations
Hematological and blood chemical investigations were performed on all non-recovery test and
control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the
lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to
necropsy on Days 29 and 43. Animals were not fasted prior to sampling.
Urinalytical investigations were performed on all non-recovery test and control group animals
during Week 4 and on all recovery group animals during Week 6. Urine samples were collected
overnight by housing the rats in metabolism cages. Animals were maintained under conditions
of normal hydration during collection but without access to food.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time
(APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11
mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing
lithium heparin anti-coagulant:
Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++), Gamma glutamyltranspeptidase.

Urinalysis
The following parameters were measured on collected urine:
Volume, Ketones, Specific Gravity, Bilirubin, pH, Urobilinogen, Protein, Blood, Glucose, Appearance.

Sacrifice and pathology:

Necropsy
On completion of the dosing period, or in the case of recovery group animals, at the end of the
treatment-free period, all animals were killed by intravenous overdose of a suitable barbiturate
agent followed by exsanguination.
At termination, excess blood from the exsanguinations procedure (after any re-bleeds and serum
sampling) was used for the production of plasma and stored frozen at approximately -70 °C. The
plasma samples were dispatched to R & D Center, 1515 State Highway #36, Union Beach, New
Jersey 07735. The subsequent fate of these samples and any investigative work performed on
them was considered to be outside of the scope of this Study Plan and is not presented in this
final report.
All animals were subjected to a full external and internal examination, and any macroscopic
abnormalities were recorded.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum
from each animal was stored frozen at approximately -20 °C. No treatment-related effects on the
pituitary-thyroid axis were identified, therefore these samples were discarded.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing
period or at the end of the treatment-free period, were dissected free from fat and weighed before
fixation:
Adrenals, Liver, Brain, Ovaries Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Pituitary (post-fixation), Thyroid/Parathyroid (post fixation), Prostate and Seminal Vesicles (with coagulating glands and fluids), Uterus with Cervix.

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10%
formalin, except where stated:
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Rectum, Salivary glands (submaxillary), Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating glands and fluids), Duodenum, Epididymides ♦, Skin (hind limb), Esophagus, Spinal cord (cervical, mid thoracic and lumbar), Eyes *, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes ♦, Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Trachea, Lungs (with bronchi)#, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus & Cervix, Mammary gland, Vagina.

* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Muscle (skeletal)
All tissues were dispatched to the histology processing Test Site Propath UK Ltd for processing (Principal Investigator: N Fower). The tissues shown below from all non-recovery control and 1200 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and kidneys from all low, intermediate and recovery dose group animals. In addition, sections of testes from all Control and 1200 ppm males were stained with Periodic Acid-Schiff (PAS) stain and examined. Microscopic examination was conducted by the Study Pathologist (Wendy Henderson).
Adrenals, Ovaries, Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating glands and fluids), Duodenum, Epididymides ♦, Spinal cord (cervical, mid thoracic and lumbar), Eyes *, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes ♦, Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Trachea, Lungs (with bronchi)#, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus & Cervix, Mammary gland, Vagina.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the Provantis™ Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann- Whitney U test (non-parametric).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Results

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

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:

Mortality
There were no unscheduled deaths.

Clinical Observations
No clinical signs of toxicity were detected.

Functional Observations
Behavioral Assessments
There were no treatment-related changes in the behavioral parameters at 300, 600 and 1200 ppm.

Functional Performance Tests
There were no toxicologically significant changes in functional performance.
Females from all treatment groups showed a statistically significant increase in the final 20% of
activity. In the absence of a true dose related response or any supporting clinical observations to
suggest an effect of neurotoxicity, the intergroup differences were considered to be of no
toxicological significance. Males treated with 1200 ppm showed a statistically significant
increase in the first test of fore limb grip strength whilst females from this treatment group
showed a statistically significant reduction in the final test of fore limb grip strength. Males
treated with 1200 ppm also showed a statistically significant reduction in overall mobility. In the
absence of any supporting clinical observations to suggest an effect of neurotoxicity, the
intergroup differences were considered to be of no toxicological significance.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.

Body Weight
Females treated with 1200 ppm showed a slight reduction in body weight gain during the first
week of treatment however statistical significance was not achieved. Recovery was evident
thereafter.
No toxicologically significant effects were detected in males treated with 1200 ppm, in animals
of either sex treated with 300 or 600 ppm or in recovery animals during the treatment free
period.
Males treated with 1200 and 600 ppm showed a statistically significant increase in body weight
gain during the second week of treatment. An increase in body weight gain is not considered to
represent an adverse effect of treatment. During the treatment free period recovery 1200 ppm
males showed a statistically significant reduction in body weight gain. The level of significance
was minimal and in the absence of a similar effect during the treatment period the intergroup
difference was considered not to be of toxicological importance.

Food Consumption
No adverse effect on food consumption or food conversion efficiency was evident in treated animals.

Water Consumption
No adverse effect on water consumption was evident in treated animals.

Laboratory Investigations
Hematology
There were no toxicologically significant effects detected in the hematological parameters examined.
Females treated with 1200 and 600 ppm showed a statistically significant reduction in
hemoglobin (p<0.05) and hematocrit (p<0.05). Although the majority of individual values were
outside of the normal ranges for rats of the strain and age used, in the absence of any associated
changes and in the absence of a true dose related response the intergroup differences were
considered of no toxicological significance. Females treated with 1200 ppm showed a
statistically significant reduction in activated partial thromboplastin time (p<0.05). The majority
of individual values were within the normal range for rats of the strain and age used therefore the
intergroup difference was considered of no toxicological importance. Recovery 1200 ppm
females showed statistically significant increases in neutrophil count (p<0.05) and reticulocyte
count (p<0.01) following fourteen days without treatment. In the absence of a similar effect
detected in non-recovery females at the end of the treatment period the intergroup differences
were considered not to be of toxicological significance.

Blood Chemistry
There were no toxicologically significant effects detected in the blood chemical parameters
examined.
Females treated with 1200 ppm showed statistically significant increases in creatinine (p<0.01)
and bilirubin (p<0.01) levels and a statistically significant reduction in aspartate
aminotransferase (p<0.05). All of the individual values were within the normal ranges for rats of
the strain and age used and therefore the intergroup differences were considered of no
toxicological importance. Recovery 1200 ppm females showed a statistically significant
increase in alkaline phosphatase (p<0.05) following fourteen days without treatment. In the
absence of a similar effect detected in non-recovery females at the end of the treatment period
the intergroup difference was considered not to be of toxicological significance. Males treated
with 300 ppm showed statistically significant reductions in alkaline phosphatase (p<0.05) and
cholesterol (p<0.05). The majority of individual values were within the normal ranges for rats of
the strain and age used and in the absence of a similar effect detected at 1200 ppm the intergroup
differences were considered of no toxicological importance.

Urinalysis
There were no treatment-related effects detected in the urinalytical parameters examined.

Pathology
Necropsy
Macroscopic necropsy findings did not indicate any adverse effect of dietary exposure at 300, 600 or 1200 ppm.
One control male had small epididymides and testes at necropsy. In the absence of treatment this was considered an isolated incident.

Organ Weights
At the end of the treatment period, males from all treatment groups showed a statistically
significant increase in liver weight both absolute and relative to terminal body weight (p<0.05-p<0.01). A true dose related response however was not evident in relative liver weight and the
majority of individual values for both absolute and relative weights were within normal ranges
for rats of the strain and age used. Microscopic examinations of liver sections only revealed
adaptive changes at 1200 ppm therefore the intergroup differences were considered not to
represent an adverse effect of treatment.
No such effects were detected in any treated female or in recovery animals following fourteen
days without treatment.
Males treated with 1200 and 600 ppm also showed a statistically significant increase in thymus
weight both absolute and relative to terminal body weight. All of the individual values were
with normal range for rats of the strain and age used and in the absence of a true dose related
response or any associated histopathological correlates the intergroup differences were
considered of not toxicological significance.

Histopathology
The following microscopic abnormalities were detected:
Liver: centrilobular hypertrophy was evident in four males and one female treated with
1200 ppm and in one recovery 1200 ppm male following fourteen days without treatment.
No such effects were detected in animals of either sex treated with 600 or 300 ppm.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The microscopic liver changes and organ weight changes identified in animals of either sex treated with 1200 ppm, were considered likely to represent adaptive changes, and were considered not to represent “serious damage” to health.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Achieved Intake

At 1200 ppm, mean achieved dosage for males was 93.2 mg/kg bw/day and for females was 98.1 mg/kg bw/day. Mean achieved intakes were fairly consistent and generally maintained the 4 fold interval between this high dietary level and the low dietary level.

At 600 ppm, mean achieved dosage for males was 46.2 mg/kg bw/day and for females was 9.1 mg/kg bw/day. Mean achieved intakes were fairly consistent and generally maintained a 2 fold interval between this intermediate dietary level and low dietary level.

At 300 ppm, mean achieved dosage for males was 22.3 mg/kg bw/day and for females was 26.2 mg/kg bw/day. Achieved intakes were generally consistent throughout the treatment period.

Results of analytical investigations

Results of accuracy analysis

Fortification (mg/g)	Concentration found (mg/g)	Recovered (%)	Mean Recovery (%)
299	299	100	100
302	299	99
1001	1034	103	103
1008	1047	103
21756	21720	100	100
21662	21650	100

 

Results of admixture analysis

Mix number	Nominal concentration (ppm)	Concentration found
		ppm	Expressed as % of nominal
1	0	ND	-
	300	299	100
	600	599	100
	1200	1195	100
2	0	ND	-
	300	295	98
	600	599	100
	1200	1206	100
3	0	ND	-
	300	305	102
	600	612	102
	1200	1225	102
4	0	ND	-
	300	294	98
	600	599	100
	1200	1188	99

Rounded results presented are based on calculations with exact data.

ND – None detected

 - Not applicable

 

Results of formulation stability analysis

Nominal concentration (ppm)	Concentration found initially (ppm)	Concentration found after Frozen storage
		Ppm	Expressed as % of initial
300	330	339	103
1000	933	874	94
20000	18770	18300	97

 

Results of formulation homogeneity analysis

Nominal concentration (ppm)	Sampling location	Concentration found (ppm)
		1	2	3	Mean
300	Side	332	332	326	330
	Middle	329	331	329	330
	Side	334	328	329	330
1000	Side	928	951	938	939
	Middle	928	931	933	931
	Side	941	922	936	933
20000	Side	18590	18820	18580	18660
	Middle	N/A	18940	18660	18800
	Side	18960	18800	18810	18860

 

Discussion

The oral administration of IFF TM11-213 to rats for a period of twenty-eight consecutive days at

dietary concentrations of 300, 600 and 1200 ppm resulted in treatment related effects detected in

animals of either sex treated with 1200 ppm.

There were no clinical signs of toxicity evident, however, females treated with 1200 ppm

showed a slight reduction in body weight gain during the first week of treatment. Initial reduced

body weight gains are often reported when the dietary admixture is unpalatable and full recovery

was evident thereafter. No adverse effects were detected in food consumption.

Histopathological examination of the liver revealed centrilobular hepatocellular hypertrophy in

four males and one female treated with 1200 ppm and in one recovery 1200 ppm male. Organ

weight data supported this finding however increases in absolute and relative liver weights were

only observed in males. The presence of centrilobular hypertrophy in one animal is of equivocal

significance however it does indicate a reversal of the change in males. Observations of this

nature are commonly observed in the rodent liver following the administration of xenobiotics

and in the absence of any degenerative or inflammatory changes is generally considered to be an

adaptive change.

There were no toxicologically significant effects observed during the weekly open field arena

observations, in the hematological/blood chemical parameters measured or at necropsy.

Applicant's summary and conclusion

Conclusions:

The oral administration of IFF TM 11-213 to rats for a period of twenty-eight consecutive days
at dietary concentrations of 300, 600 and 1200 ppm resulted in treatment related effects detected
in animals of either sex treated with 1200 ppm.
The effect on body weight gain in females at 1200 ppm was considered to reflect the initial
reluctance to eat the dietary formulation and not to represent an adverse effect of treatment. The
microscopic liver changes and organ weight changes identified in animals of either sex treated
with 1200 ppm, were considered likely to represent adaptive changes, and were considered not to
represent “serious damage” to health.
For these reasons 1200 ppm may be regarded as a “No Observed Adverse Effect Level”
(NOAEL) for animals of either sex.

Executive summary:

The repeated dose toxicity of the test substance, TM 11-213, was assessed according to OECD Test Guideline 407 using a continuous dietary admixture method. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 1200 ppm for males and females.