2-Butenoic acid, 1-ethyl-2-methylpropyl ester, (2E)-

Endpoint summary

Description of key information

The repeated dose toxicity of the test substance, TM 12-209, was assessed in rats according to OECD Test Guideline 407. The test item was administered by continuous dietary admixture to rats, for twenty-eight consecutive days, at dietary concentrations of 500, 3500 and 15000 ppm (equivalent to a mean achieved dosage of 26.4, 215.1 and 1010.7 mg/kg bw/day for males and 28.5, 254.3 and 1110.3 mg/kg bw/day for females respectively). The control group was treated with basal laboratory diet. Two recovery groups were treated with the high dose (15000 ppm) or basal laboratory diet for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Each group had five males and five females. Clinical signs, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated at the end of treatment. All animals were subjected to gross necropsy examination and histopathological examination of selected tissues was performed. 
The microscopic changes in the prostate, seminal vesicles and coagulating glands and the associated changes in the weight of these organs in males treated with 15000 ppm were considered to represent an adverse effect of treatment. Additionally, granular casts and corticomedullary tubular basophilia in the kidneys of males treated with 15000 ppm were considered to indicate an effect of nephrotoxicity and therefore also represent an adverse effect of treatment. Hyaline droplet formation was observed in the kidneys of males from control, treatment and recovery groups but this effect is a species and sex specific condition in rats and therefore normally does not represent a risk to humans. For these reasons, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 3500 ppm for males (equivalent to 215.1 mg/kg bw/day) and 15000 ppm for females (equivalent to 1110.3 mg/kg bw/day). 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 July 2014 and 04 June 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.

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

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 seven 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 196 to 232g, the females
weighed 150 to 179g, 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.

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 500,
3500 and 15000 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 H800 mixer.

The stability and uniformity of distribution of the test item in the diet were determined by Harlan
Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study. Results showed the
dietary admixtures at 500 ppm to be stable for three weeks when stored frozen. Analysis of
dietary admixtures at 1000 and 15000 ppm showed the admixtures to be stable for 6 weeks at
room temperature. Dietary admixtures were prepared prior to the first treatment, and weekly
thereafter. The 500 ppm diet was stored in labelled, double plastic bags in the freezer. The 3500
and 15000 ppm diet was stored in labelled, double plastic bags in labelled, covered plastic bins at
room temperature. Samples were taken from the dietary admixtures and analysed for uniformity
of distribution and concentration at Harlan Laboratories Ltd, Shardlow, UK, Analytical Services.
The method used for analysis of formulations and the results obtained are given in Appendix 20.
The results indicate that the mean prepared dietary admixture concentrations were generally
within acceptable ranges for the purpose of this study. Analysis of the concentration of Test
Item in the dietary admixtures at 500 ppm during the first two weeks of the study revealed that
the concentrations of IFF TM 12-209 in the dietary admixtures were lower than expected. It is
believed the lower concentrations observed were a result of loss of the test item during
preparation of the dietary pre-mix due to the small amount of test item used. The method of
preparation of the dietary admixtures for the 500 ppm concentration was subsequently changed
in order to increase the level of test item in the admixture. The final two dietary admixtures were
prepared by taking a known amount of the prepared 15000 ppm dietary and mixing with an
appropriate amount of basal laboratory diet to achieve the 500 ppm dietary 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 samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.

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

Analysis of sample
The dietary admixtures received were extracted with methanol. An aliquot of dietary admixture was accurately weighed into a suitable jar and left to extract overnight in ambient conditions. The sample solutions were then ultra-sonicated for 30 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with methanol 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. There samples were then prepared for analysis as the test samples.

Preparation of linearity standards
A range of standard solutions were prepared in methanol from a stock solution of 1010 ppm by serial dilution covering the concentration range 0 to 202 ppm.

Instrument setup
GC system: Agilent Technologies 5890, incorporating autosampler and workstation.
Column: ZB-5 (30 m x 0.53 mm id x 5 µm film).
Oven temperature programme: Oven: 50 °C, for 0 minutes, with 10 °C/minute to 170 °C for 0 minutes then 50 °C/minute to 300 °C for 5 minutes.
Injection temperature: 250 °C
Flame ionisation detector temperature: 250 °C
Injection volume: 1 µL
Retention time: ~ 11 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 methods
Specificity
The control diet samples and an analysed solvent blank showed no significant interfering response at the retention time of the test item. The standard solutions contained a peak specific for hte 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 within the calibration range. The R2 fit of the calibration curve for the data was 1.0000 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 55 % to 103 %.
The test item was found to be stable in the admixtures when kept for 6 weeks (1000 and 15000 ppm) at room temperature and 3 weeks (500 ppm) frozen due to results which met the variation limit of 20 % from the time-zero mean.
The admixtures 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:

The test item was administered continuously, for twenty-eight consecutive days, by dietary admixture, followed by a 14 day recovery period.

Frequency of treatment:

The test item was administered continuously, for twenty-eight consecutive days, by dietary admixture.
During the recovery period, all animals were given untreated diet.

Remarks:

Doses / Concentrations:
0, 500, 3500, 15000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

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

Control animals:

yes, plain diet

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 26, 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.

Estrous Cycle
A vaginal smear was taken daily for non-recovery females during Weeks 3 and 4 and for recovery females throughout the treatment free period (see deviations to Study Plan). The sample was placed on a glass slide and the smears were allowed to dry and then stained using a diluted giemsa stain. The slides were examined microscopically and the stage of estrus was recorded.

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.

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.
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, Muscle (skeletal).
♦ Preserved in Modified Davidson’s fluid
# Eyes fixed in Davidson’s fluid

All tissues were dispatched to the histology processing Test Site Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU for processing (Principal Investigator: N Fower).
The tissues listed below from all non-recovery control and 15000 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:
Adrenals, Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and Pons, Caecum, Colon, Duodenum, Epididymides, Eyes, Ovaries, Pituitary, Prostate, Rectum, Sciatic nerve, Seminal vesicles (with coagulating glands and fluids), Spinal cord (cervical, mid thoracic and lumbar), 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, Muscle (skeletal).

Any macroscopically observed lesions were also processed together with the liver and kidney from all 500 and 3500 ppm dose group animals. In addition, sections of testes from all Control and 15000 ppm males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Since there were indications of treatment-related prostate, seminal vesicle, coagulating gland and kidney changes in males, examination was subsequently extended to include similarly prepared sections of prostate, seminal vesicle, coagulating gland and kidney from males in the low and intermediate groups (see deviations to Study Plan).
Microscopic examination was conducted by the Study Pathologist (J Wilson at Propath GmbH, Muttenzerstrasse 30, 4133 Pratteln, Switzerland). A peer review of the histopathology examinations was also performed by W Henderson.

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):

effects observed, treatment-related

Description (incidence and severity):

See results

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

See results

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

no effects observed

Ophthalmological findings:

not examined

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:

effects observed, treatment-related

Description (incidence and severity):

See results

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
There were no clinical signs of toxicity detected.

Functional Observations
Behavioral Assessments
There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests
There were no toxicologically significant changes in functional performance.
All treated females had a statistically significant reduction in overall motor activity when
compared to controls. Females treated with 500 ppm also had a statistically significant reduction
in the last 20% of mobile activity. All individual parameters were within normal background
ranges and in the absence of any clinical observations to suggest an effect of neurotoxicity, the
intergroup differences were considered not to be toxicologically significant.

Sensory Reactivity Assessments
There were no toxicologically significant changes in sensory reactivity. All inter and intra group
differences in sensory reactivity scores were considered to be a result of normal variation of rats
of the strain and age used and were of no toxicological importance.

Body Weight
Group mean body weight gains for males treated with 15000 ppm were slightly lower than
controls for the duration of the treatment period; differences from control achieved statistical
significance during the first week of treatment. Recovery was evident during the treatment-free
period where body weight gains were similar to controls.
Females treated with 15000 ppm had statistically significantly lower body weight gains during
the first week of treatment when compared to controls. Recovery was evident thereafter.
No such effects were detected in animals of either sex treated with 500 or 3500 ppm.
Body weight gains for males treated with 500 or 3500 ppm were lower than controls during the
final week of treatment, however, statistical significance was not achieved, and therefore, the
intergroup differences were considered to represent normal biological variation.

Food Consumption
Males treated with 15000 ppm had slightly lower food consumption throughout the treatment
period when compared to controls. Females treated with 15000 ppm had slightly lower food
consumption during the first week of treatment when compared to controls. No such effects were
detected in animals of either sex treated with 500 or 3500 ppm. Food consumption throughout
the recovery period was comparable to controls.
Food efficiency for animals of either sex treated with 15000 ppm was lower than controls during
the first week of treatment. This was consistent with the intergroup differences in body weight
gain and food consumption observed.

Water Consumption
No obvious effect on water consumption was detected for any treated animal.

Estrous Cycle Assessments
One recovery control female was acyclic. All remaining main phase and recovery females
showed evidence of estrus and were therefore considered to have a regular estrous cycle.

Laboratory Investigations
Hematology
There were no toxicologically significant changes in any of the hematological parameters
measured.
At the end of the treatment period, females treated with 15000 ppm had a statistically significant
reduction in mean corpuscular haemoglobin concentration. Females treated with 500 ppm had a
statistically significant increase in neutrophil count. The majority of individual parameters were within normal background ranges and in the absence of any histopathological correlates, the
intergroup differences were considered not to be of toxicological importance.

Blood Chemistry
There were no toxicologically significant changes in the blood chemistry parameters measured.
At the end of the treatment period, males treated with 15000 ppm had a statistically significant
increase in albumin levels. All individual parameters were within normal background ranges
therefore this intergroup difference was considered to be of no toxicological importance.
Females treated with 3500 ppm had a statistically significant increase in γ-glutamyl
transpeptidase. Three out of five of the individual measurements for this parameter were above
the upper limit of the normal ranges, however, in the absence of a true dose related response and
in the absence of any histopathological correlates in the liver, the intergroup difference was
considered not to be toxicologically significant.
At the end of the treatment-free period, males treated with 15000 ppm had a statistically
significant reduction in cholesterol levels. In the absence of any histopathological correlates in
the liver, or similar reduction at the end of treatment this intergroup difference was considered to
be of no toxicological significance.

Urinalysis
There were no toxicologically significant effects detected in the urinalytical parameters
examined.
Following the two week recovery period, urine volume for males was statistically significantly
higher than controls. Specific gravity of the urine for these males was also lower than control
with differences attaining statistical significance. In the absence of a similar effect detected at
the end of the treatment period, the intergroup differences were considered to be of minimal
toxicological significance.

Pathology
Necropsy
Two males treated with 15000 ppm had small prostate and seminal vesicles.
No such effects were detected in males treated with 500 or 3500 ppm. No macroscopic
abnormalities were detected for any treated female.
One control female had reddened mandibular lymph nodes. In the absence of treatment, this
finding was considered to be incidental.

Organ Weights
At the end of the treatment period, males treated with 15000 ppm had a statistically significant
reduction in prostate and seminal vesicle weight.
No such effects were detected in males treated with 500 or 3500 ppm or following the two week
recovery period for males treated with 15000 ppm.
At the end of the treatment period, males treated with 15000 ppm had a statistically significant
reduction in absolute liver weight and a statistically significant increase in liver weight relative
to terminal body weight. Females treated with 15000 ppm had a statistically significant increase
in liver weight, both absolute and relative to terminal body weight and a statistically significant
reduction in thyroid weight, both absolute and relative to terminal body weight, and at the end of
the treatment free period, females continued to show a statistically significant increase in liver
weight, both absolute and relative to terminal body weight. The majority of individual values for
these organs were within normal background ranges and in the absence of any supporting
histopathology in the liver or thyroid, the intergroup differences were considered not to be
toxicologically significant.

Histopathology
Microscopic alterations were noted in the kidneys, prostate, seminal vesicles and coagulating
glands of Group 4 males and in the kidneys of Group 3 males. These consisted of:

Kidneys: Granular casts were present at slight degree in two group 4 males and
corticomedullary tubular basophilia was recorded at slightly increased incidence and severity (up
to slight) in all five males of Group 4. Cortical hyaline droplets were present at increased
severity up to moderate in all Group 3 and Group 4 males.
Prostate Gland, Seminal Vesicles and Coagulating Glands: Hypoplastic acinar epithelium
was noted at minimal to moderate degree in three animals.
Following the fourteen day treatment-free recovery phase the following treatment related
findings were recorded in Group 4 males:
Kidneys: Granular casts at minimal degree were noted in one instance. Corticomedullary tubular
basophilia remained at slightly increased incidence and severity (up to slight) in four animals.
Cortical hyaline droplets were present at minimal to moderate severity in all 5 males.

Dose descriptor:

NOAEL

Effect level:

215 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

1 110 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Critical effects observed:

not specified

Achieved Intake

At 15000 ppm, mean achieved dosage for males was 1010.7 mg/kg bw/day. Mean achieved intake became progressively lower throughout the study period, however, an approximate fourfold interval was maintained between the high dietary level and the intermediate dietary level. Mean achieved dosage for females was 1110.3 mg/kg bw/day. Mean achieved intake was fairly consistent for the first three weeks of the study; during the final week, mean achieved intake was slightly lower. A four-fold interval between the high dietary level and the intermediate dietary level was generally maintained. At 3500 ppm, mean achieved dosage for males was 215.1 mg/kg bw/day. Mean achieved intake was fairly consistent during the first three weeks of the study; during the final week mean achieved intake was slightly lower. Mean achieved dosage for females was 254.3 mg/kg bw/day. Mean achieve intake was fairly consistent throughout the treatment period. At 500 ppm, mean achieved dosage for males was 26.4 mg/kg bw/day. Mean achieved intake was lower during Week 2 of the study however mean achieved intake for the rest of the study was relatively consistent. Mean achieved dosage for females was 28.5 mg/kg bw/day. Mean achieved intake during the first two weeks was slightly lower than in subsequent weeks.

Discussion

The oral administration of IFF TM 12-209 to rats for a period of twenty-eight consecutive days at dietary concentrations of 500, 3500 and 15000 ppm resulted in treatment-related effects in males treated with 15000 ppm. No clinical signs of toxicity were apparent throughout the study. Body weight gains for animals of either sex treated with 15000 ppm were statistically significantly lower during the first week of treatment with an associated reduction on food consumption. This reduction is likely to reflect an initial distaste to the treated diet rather than an effect of systemic toxicity. Body weight gain and food consumption for females thereafter was similar to controls, however males continued to show slightly lower body weight gain and food consumption throughout the treatment period, albeit to a lesser extent than during the first week of treatment. At the end of the treatment period, males treated with 15000 ppm had a statistically significant reduction in prostate and seminal vesicle weight. Microscopic examination of the prostate, seminal vesicles and coagulating gland revealed hypoplastic acinar epithelium in three males at minimal to moderate severity. No such effects were detected in males previously given this dietary concentration following the fourteen day recovery period. Despite the effects in the prostate, seminal vesicles and coagulating gland appearing reversible, the findings in the nonrecovery males were considered to represent an adverse effect of treatment, however the aetiology of the effect is unclear, especially when considering that no effects were seen in the testis/epididymis that suggest an effect upon reproductive capability.

Microscopic examination of the kidneys revealed minimal or slight corticomedullary tubular basophilia in all non-recovery males treated with 15000 ppm; granular casts were also present at slight degree in two of these males. Following the fourteen day recovery period, corticomedullary tubular basophilia was present at slightly increased incidence and severity in four out of five males previously treated with 15000 ppm. Granular casts were present at minimal degree in one male previously treated with 15000 ppm. The presence of granular casts in association with corticomedullary tubular basophilia are indicative of nephrotoxicity, and therefore these findings represent an adverse effect of treatment. At the end of the treatment free period, males previously treated with 15000 ppm had statistically significantly lower urine specific gravity and statistically significantly higher urine volume. Despite no similar changes in urine volume or specific gravity being apparent at the end of treatment, an association with treatment cannot be discounted due to the microscopic findings apparent in the kidneys. Cortical hyaline droplets were recorded at increased severity up to moderate in all males treated with 3500 ppm and in males treated with 15000 ppm at the end of treatment and following the fourteen day recovery period. Hyaline droplet formation within the tubules is consistent with the accumulation of alpha 2u-globulin, which is specific to the male rat and does not represent a risk to humans. A range of chemicals are known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury, the formation of granular casts and increased cell turnover as manifested by tubular basophilia. Although the presence of corticomedullary tubular basophilia and granular casts in the kidneys of males treated with 15000 ppm were considered to represent an adverse effect of treatment, these findings may be a consequence of the increased incidence and severity of hyaline droplet formation. Lower concentrations of the test item in the dietary admixtures at 500 ppm during the first two weeks of treatment were considered not to have impacted on the integrity of the study.

Conclusions:

The dietary oral administration of IFF TM 12-209 to rats for a period of twenty-eight consecutive days at concentrations of 500, 3500 and 15000 ppm resulted in treatment-related effects in males treated with 3500 and 15000 ppm. Granular casts and corticomedullary tubular basophilia in the kidneys of males treated with 15000 ppm were considered to indicate an effect of nephrotoxicity and therefore were considered to represent an adverse effect of treatment. Hyaline droplet formation in the kidneys of males treated with 3500 ppm was not associated with any degenerative kidney changes; in terms of extrapolation to man and risk assessment calculations, this effect is species and sex specific and therefore does not represent a risk to humans. The microscopic changes in the prostate, seminal vesicles and coagulating glands and the associated changes in the weight of these organs in males treated with 15000 ppm were considered to represent an adverse effect of treatment. For these reasons, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 3500 ppm for males and 15000 ppm for females.

Executive summary:

The repeated dose toxicity of the test substance, TM 12-209, was assessed in rats according to OECD Test Guideline 407 using a continuous dietary admixture method. The test item was administered by continuous dietary admixture to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dietary concentrations of 500, 3500 and 15000 ppm (equivalent to a mean achieved dosage of 26.4, 215.1 and 1010.7 mg/kg bw/day for males and 28.5, 254.3 and 1110.3 mg/kg bw/day for females respectively). A control group of five males and five females were treated with basal laboratory diet. Two recovery groups, each of five males and five females, were treated with the high dose (15000 ppm) or basal laboratory diet for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological examination of selected tissues was performed.

The microscopic changes in the prostate, seminal vesicles and coagulating glands and the associated changes in the weight of these organs in males treated with 15000 ppm were considered to represent an adverse effect of treatment. Additionally, granular casts and corticomedullary tubular basophilia in the kidneys of males treated with 15000 ppm were considered to indicate an effect of nephrotoxicity and therefore also represent an adverse effect of treatment. Hyaline droplet formation was observed in the kidneys of males from control, treatment and recovery groups but this effect is a species and sex specific condition in rats and therefore normally does not represent a risk to humans. For these reasons, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 3500 ppm for males (equivalent to 215.1 mg/kg bw/day) and 15000 ppm for females (equivalent to 1110.3 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

215 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The repeated dose toxicity of the test substance, TM 12-209, was assessed in rats according to OECD Test Guideline 407 (Harlan, 2015). The test item was administered by continuous dietary admixture to rats, for twenty-eight consecutive days, at dietary concentrations of 500, 3500 and 15000 ppm (equivalent to a mean achieved dosage of 26.4, 215.1 and 1010.7 mg/kg bw/day for males and 28.5, 254.3 and 1110.3 mg/kg bw/day for females respectively). Treatment related effects detected in males treated with 3500 and 15000 ppm. However those findings do not warrant a STOT 2 classification, because the following reasons:

In the OECD 407 study, granular casts and increased corticomedullary tubular basophilia observed in the kidneys of males treated with 15000 ppm were considered to indicate an effect of nephrotoxicity and represent an adverse effect of treatment. However a relationship with hyaline droplet formation cannot be fully discounted, since a range of chemicals are known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury, the formation of granular casts and increased cell turnover as manifest by tubular basophilia. The relationship was further confirmed in the OECD 421 study, as an increase in incidence and severity of hyaline droplets and corticomedullary tubular basophilia was evident in males from all treatment groups and therefore the kidney findings in males treated with 15000 ppm, consisting of granular casts was considered to be correlated to the finding of hyaline droplets, which is species and sex specific, not relevant to human. Therefore we believe the kidney effects seen in both studies were findings related to alpha 2u-globulin nephropathy.

 

In terms of the microscopic changes in the prostate, seminal vesicles and coagulating glands and the associated changes in the weight of these organs in males treated with 15000 ppm in the OECD 407 study, those were not confirmed in the follow-up OECD 421 study. In the OECD 421 study males treated with 15000 and 3500 ppm showed a statistically significant (p<0.05) reduction in prostate weight both absolute and relative to terminal body weight, however in the absence of a true dose related response or any associated histopathological correlates the intergroup differences were considered not to be of toxicological importance.

 

The OECD 421 study provides a much more powerful/comprehensive assessment of potential effects of the test substance TM 12-209 at levels up to and including 15000 ppm overall on males because (1) a larger group size has been employed; (2) males were treated for 8 weeks of before termination; (3) a comprehensive range of endpoints of the performance and integrity of the males reproductive system have been evaluated. In the OECD 421 study,a ‘No ObservedAdverse Effect Level’ (NOAEL) for males can be established at the high dose (15000 ppm) excluding those findings related to alpha 2u-globulin nephropathy.

 

For the above reasons and weight of evidence, the test substance is not classified for specific target organ toxicity following repeated exposure.   

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
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

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 3500 ppm (215.1 mg/kg bw/day) for males and 15000 ppm (1110.3 mg/kg bw/day) for females in an OECD 407 study, due to the observed granular casts and corticomedullary tubular basophilia in the kidneys of males treated with 15000 ppm and the microscopic changes in the prostate, seminal vesicles and coagulating glands and the associated changes in the weight of these organs in males treated with 15000 ppm in the 28 day dietary study. However the above described findings in prostate were not seen in a follow-up OECD 421 study. In addition the kidney effects were more believed to related to 2u-globulin nephropathy based on the finding in the OECD 421 study. Therefore the NOAEL for male systemic toxicity can be considered to be the high dose, 15000 ppm. Together, hazard classification is not warranted for this material from repeated dose toxicity perspective, according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.