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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Rabbits were administered the test substance, and the vehicle control (corn oil) formulations orally via gavage once daily from Day 1 to Day 14.
This study was performed in 2011 in a GLP facility in accordance with OECD guidelines and good scientific practice. It was reported, however, as being non-GLP and cannot be verified as being conducted to full GLP standards. Based on the evidence available the study is considered to be acceptable as a key study as it has been conducted in a GLP facility and in accordance with principles essentially equivalent to GLP standards.
GLP compliance:
yes
Remarks:
2011 GLP facility Study in accordance with OECD guidelines and good scientific practice. Study reported as non-GLP, full GLP standards not verified. Acceptable as key study conducted in GLP facility in accordance with principles equivalent to GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Test Substance
Identification: Cyclamen Aldehyde
Batch (Lot) No.: VE00130506
Receipt Date: 27 Jan 2011
Expiration Date: 04 Mar 2012
Physical Description: Clear, colorless liquid
Purity: Purity assumed to be 100% for dose calculation purposes.
Storage Conditions: Room temperature, protected from light
Supplier: Givaudan Suisse SA 5, Chemin de la Parfumerie, Vernier, Switzerland

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male
Details on test animals and environmental conditions:
Receipt
Twenty-one male Hra:(NZW)SPF rabbits were received from Covance Research Products, Inc., Swampbridge Road, Box 7200, Denver, PA. Body weight range was 2.3 to 3.0 kg on the day ofarrival, and was 2.4 to 3.1 kg at randomization. The rabbits were approximately 5 months of age at arrival to the Testing Facility.

Justification for Test System and Number of Animals
The New Zealand White [Hra:(NZW)SPF] rabbit was selected as the Test System because it is one nonrodent mammalian species accepted and widely used throughout the industry. The total number of animals used in this study was the minimum required to properly characterize the effects of the test substance. In addition, this study did not duplicate any previous work.

Animal Identification
Rabbits were permanently identified using Monel® self-piercing ear tags. Male rabbits were given unique permanent identification numbers when assigned to the study.

Environmental Acclimation
After receipt at the Testing Facility, the rabbits were acclimated for at least 1 day prior to predose sperm sample collection.

Selection, Assignment, and Replacement of Animals
Upon arrival, rabbits were assigned to individual housing on the basis of computer-generated random units. After acclimation, rabbits were selected for study on the basis of physical appearance and body weights recorded during acclimation. The rabbits were assigned to 4 dose groups (Groups 1 through 4), 5 rabbits per dose group based on computer-generated (weightordered) randomization procedures.

Disposition
The remaining rabbit not assigned to study was humanely euthanized.

Husbandry
Housing
The rabbits were individually housed in units of six to eight stainless steel cages. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals. Cage pan liners were changed at least three times weekly. Cages were changed approximately every other week.

Environmental Conditions
The study rooms were maintained under conditions of positive airflow relative to a hallway and independently supplied with a minimum of 10 changes per hour of 100% fresh air that had been passed through 99.97% HEPA filters. Room temperature and humidity were monitored constantly throughout the study. Room temperature was targeted at 61°F to 72°F (16°C to 22°C); relative humidity was targeted at 30% to 70%.
An automatically controlled 12-hours light:12-hours dark fluorescent light cycle was maintained. Each dark period began at 1900 hours (± 30 minutes).

Food
Approximately 150 g of Certified Rabbit Chow® #5322 (PMI® Nutrition International, St. Louis, MO) was available to each rabbit each day. The certified food was available from individual, stainless steel, "J-type" feeders attached to each cage.
Analyses were routinely performed by the food supplier. No contaminants at levels exceeding the maximum concentration limits for certified food or deviations from expected nutritional requirements were detected by these analyses. Copies of the results of the food analyses are available in the raw data.
Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to have been present in the food that would have interfered with the results of this study.

Water
Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rabbits ad libitum from an automatic watering access system. Chlorine was added to the processed water as a bacteriostat.
The processed water is analyzed twice annually for possible chemical contamination (Lancaster Laboratories, Lancaster, PA) and monthly for possible bacterial contamination (QC Laboratories, Southampton, PA). Copies of the results of the water analyses are available in the raw data.
Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to have been present in the water that would have interfered with the results of this study.

Animal Enrichment
For psychological enrichment, rabbits were provided with items such as a bell or rattle. Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to have been present in the chewable enrichment devices at levels that would have interfered with the results of this study.

Veterinary Care
Upon animal arrival and twice during the course of the study, rabbits were examined by the veterinary staff. Records of examinations are maintained with the raw data. No medical treatments were administered. None of the medical examinations had an adverse impact on the integrity of the study data or on the interpretation of the study results.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Rabbits were administered the test substance and/or the control article formulations orally (stomach tube) once daily on Day 1 of study (DS 1) to 14. The dose volume for each animal was based on the most recent body weight measurement. The first day of dosing for each animal was
designated as DS 1. The dosing formulations were stirred continuously during dose administration.

Justification of Route and Dosage Levels
The oral gavage route was selected for use to maximize systemic absorption. Dosage levels were chosen based on a comparative study made on 4-tert butyl priopionaldehyde, a structurally similar substance, which was tolerated with no acute toxicity at up to 300 mg/kg in the rabbit and shows equivalent acute toxicity potential to cyclamen aldehyde in rats.
Duration of treatment / exposure:
once daily doses for 14 consecutive days
Frequency of treatment:
once daily doses for 14 consecutive days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
In-life Procedures, Observations, and Measurements
The in-life procedures, observations, and measurements listed below were performed for all rabbits.

Viability Checks
The rabbits were assessed for viability at least twice daily during the study.

General Appearance
The rabbits were observed for general appearance daily during the predose period, before each dose was administered and on the day of scheduled euthanasia.

Postdose Observations
For the first 4 days of dosing, postdose observations were conducted immediately after each animal was administered the test substance or control article, then at hourly intervals for the first four hours after dose administration and again at the end of the normal working day. Beginning on the fifth day of dosing, postdose observations were conducted between 1 to 2 hours after dose
administration and at the end of the normal working day.

Body Weights
Body weights were recorded on the day of arrival at the Testing Facility, at least once during the predose period, daily during the dose period, and on the day of scheduled euthanasia.

Food Consumption
Food consumption values were recorded daily after arrival at the Testing Facility, daily during the dose period, and prior to placement of the rabbits in metabolism cages (food left value).

Laboratory Evaluations
Urinalysis
Urine samples (as much as possible) were collected overnight from all rabbits once on DS 14. Food was withheld during the collection interval. Overnight urine samples were collected over cold packs. For 1, 2, 2, and 1 in each of the 4 respective dose groups, urine samples were unable to be obtained overnight; therefore, a urine sample was collected from the urinary bladder following euthanasia using a 21 G needle. For these rabbits (8582 in the 0 mg/kg/day dose group, 8584 and 8586 in the 30 mg/kg/day dose group, 8588 and 8589 in the 100 mg/kg/day dose group and 8596 in the 300 mg/kg/day dose group) , the 21 G needle was inserted into the urinary bladder and as much urine as possible was collected into an appropriately sized syringe. Urine samples were stored in appropriately sized, labeled polypropylene tubes and then stored in a freezer set to maintain -20°C or colder for possible future analysis. Storage tubes were labeled at minimum with the Testing Facility study number, rabbit number, group number, dosage level, day of study, date of collection, species, biological matrix and storage conditions.

Male Reproductive Assessments
Sperm Evaluation
Semen samples were collected from each rabbit prior to initiation of dose administration and on the day of scheduled euthanasia (DS 15). Predose sample collections were attempted on four days prior to initiation of dosage administration. Not all rabbits yielded a sample or a yielded a viable sample at each predose collection day. Predose data will be retained in the raw data and will not be summarized in this report. Samples were collected utilizing an artificial vagina (and a teaser female rabbit of the same source and strain), and analyzed for motility and sperm count utilizing the Hamilton Thorne IVOS for computer-assisted sperm analysis (CASA) or manually for sperm morphology.

Sperm Motility
Sperm motility was evaluated following dispersion, into an appropriate medium, of semen ejaculate.

Sperm Count
Sperm count was evaluated following dispersion, into an appropriate medium, of semen ejaculate.

Sperm Morphology
Sperm morphology was evaluated from a stained sperm smear prepared from a semen ejaculate. Due to an insufficient volume of ejaculate collected prior to euthanasia, the sperm morphology was evaluated from the left cauda epididymis collected after euthanasia. Four slides were prepared, and approximately 200 sperm cells were evaluated from each animal. Images of motility and concentration samples were taken, retained as electronic images and archived with the raw data.


Sacrifice and pathology:
Scheduled Euthanasia and Necropsy
On DS 15 (the day following the completion of the 14-day dose period), rabbits were euthanized via an intravenous injection of a euthanasia solution (390 mg pentobarbital sodium and 50 mg phenytoin sodium) and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. See Section 6.12.3. (Tissue Collection and Preservation) for tissues retained, weighed and evaluated microscopically. For 6 rabbits, urine samples were collected from the urinary bladder following euthanasia using a 21 G needle as described in Section 6.11.1. (Urinalysis).
Organ Weights
The organs identified below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, where required. Organ to body weight ratio (using the terminal body weight) were calculated.
Organs weighed at necropsy:
Epididymis - Individually weighted.
Gland, prostate
Gland, seminal vesicle (with and without fluid)
Liver (gallbladder drained prior to weighing)
Kidney - Paired organ weight.
Testis - Paired organ weight. Fixed in Bouin’s solution for 48 to 96 hours, rinsed per Testing Facility Standard Operating Procedures, and
then preserved in Modified Davidson’s fixative; individually weighed.

Tissue Collection and Preservation
Representative samples of the tissues identified below were collected from all rabbits and preserved in 10% neutral buffered formalin, unless otherwise indicated.
Epididymides
Epididymis, left cauda - Retained due to an insufficient volume of semen ejaculate collected for the preparation of sperm morphology sample analysis
Gland, prostate
Gland, seminal vesicle
Kidney
Liver
Testes - Fixed in Bouin’s solution for 48 to 96 hours, rinsed per Testing Facility Standard Operating Procedures, and then preserved in Modified Davidson’s fixative.

Histology
Tissues were processed at Charles River Laboratories Pathology Associates - Maryland. Tissue trimming was performed at the Testing Facility. All other histology procedures were performed by Charles River Laboratories Pathology Associates - Maryland. The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
• The testes and epididymides from each rabbit in Groups 1 and 4
Histopathology
Histopathological evaluation was performed by a board-certified veterinary pathologist. The following tissues were evaluated microscopically:
• The testes and epididymides from each rabbit in Groups 1 and 4
Statistics:
Means and standard deviations were calculated for body weights, food consumption, sperm count and motility and organ weights.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Mortality
All rabbits survived to scheduled euthanasia.

Clinical Observations
None of the clinical signs that occurred were attributed to administration of cyclamen aldehyde because they were transient and were limited to one rabbit in the 100 or 300 mg/kg/day dosage groups. These clinical signs included scant feces and no feces in the cage pan. No other clinical signs occurred.

Body Weights and Body Weight Changes
Body weights and body weight gains were unaffected by dosages of cyclamen aldehyde as high as 300 mg/kg/day. Overall, body weights and body weight gains were comparable among the dosage groups for the cumulative dosage period (DSs 1 to 14). All groups, including the control group, lost body weight between DS 14 and DS 15 because of overnight fasting for urine collection.
Body weight gains in the 30, 100 and 300 mg//kg/day dosage groups were 108%, 119% and 115% of the control group value, respectively, for the cumulative dosage period (DSs 1 to 14). The average body weight on DS 14 (day of last dosage) was 101%, 99% and 100% of the control group value in the 30, 100 and 300 mg/kg/day dosage groups, respectively.

Food Consumption
Absolute (g/day) and relative (g/kg/day) food consumption values were unaffected by dosages of cyclamen aldehyde as high as 300 mg/kg/day. Food consumption values were also comparable among the dosage groups for the cumulative dosage period. Absolute food consumption values in the 30, 100 and 300 mg//kg/day dosage groups were 102%, 100% and 103% of the control group value, respectively, for the cumulative dosage period (DSs 1 to 14). Relative food consumption values were 101%, 102% and 103% of the control group value in the 3 respective cyclamen aldehyde-treated groups during the same period.

Gross Pathology
There were no gross lesions observed at necropsy examination. All rabbits appeared normal.

Organ Weights
Terminal body weights were comparable among the four dosage groups. The weights of the epididymides, left cauda epididymis, testes, seminal vesicles (with and without fluid) and prostate and the ratios of these organ weights to terminal body weight were unaffected by dosages cyclamen aldehyde as high as 300 mg/kg/day. In addition, there were no test substance-related changes in the non-reproductive organs (i.e., liver or paired kidneys) at any dosage level. There were no patterns, trends, or correlating data to suggest these values were toxicologically relevant. The apparent increase in the absolute and relative (% body weight) weights of the liver that occurred at 300 mg/kg/day (18% and 21% over controls, respectively) was largely attributed to one rabbit (no. 8596) in the 300 mg/kg/day dosage group with an absolute liver weight of 111.1 g (range: 70.1 to 92.3 for other rabbits in the group). Exclusion of this rabbit from summarization would still result in a higher group mean weight in comparison to the control group value. The absolute and relative (% body weight) weights of the paired kidneys were also increased in the 300 mg/kg/day dosage group (11% and 13% over controls, respectively), again attributed to one rabbit (no. 8595) in this dosage group. Overall, the individual organ weight differences observed were considered incidental and unrelated to administration of cyclamen aldehyde.

Sperm Evaluations
The values for the number of motile sperm and total sperm count from ejaculated semen samples were highly variable across the dosage groups, including the control group. Whilst a slight trend in the mean number of motile sperm (596.6, 543.3 and 431.0 in the 30, 100 and 300 mg/kg/day dosage groups, respectively, vs. 627.0 in controls) and total sperm count (616.6, 595.5 and 496.2 in the 30, 100 and 300 mg/kg/day dosage groups, respectively, vs. 679.0 in controls) from ejaculated samples in the cyclamen aldehyde-treated groups was observed, individual values were highly variable and the lowest reported individual values were within the range of the control group values. In general, all values across all treated groups were within the range of the concurrent control group values and/or the historical control range at the Testing Facility. The observed trend in sperm motility and total sperm count was not considered an adverse finding of Cyclamen Aldehyde. Values for percent motile sperm, number of nonmotile sperm from the semen ejaculate sample and cauda epididymal sperm count and density were comparable among the four dosage groups. In addition, there were no patterns or trends in the morphology data to suggest any toxicological relevance.

Histopathology
No test substance-related microscopic changes were observed in the testes or epididymides examined.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
Remarks on result:
other: n/a

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The objective of this study was to determine the potential toxicity of Cyclamen Aldehyde, when given orally via stomach tube for 14 consecutive days to male New Zealand White rabbits. In addition, potential effects on the male reproductive organs, including sperm parameters were assessed.
In the current study, Cyclamen Aldehyde at doses as high as 300 mg/kg/day did not result in any mortality or gross or microscopic lesions, and did not increase the incidence of clinical signs in the male rabbits. There were no apparent effects on body weight, body weight gains, food consumption or reproductive and non-reproductive organ weights. Cyclamen Aldehyde did not affect the cauda epididymal sperm count or sperm morphology. Individual sperm motility results, specifically the number of motile sperm and the total count of sperm from ejaculated semen samples, were highly variable but there were no noteworthy changes observed in comparison with concurrent and/or historical control values.
In conclusion, Cyclamen Aldehyde when administered orally to male rabbits for 14 days at dosages as high as 300 mg/kg/day did not produce any clinical observations, changes in body weight or food consumption or affect the weights of any reproductive or non-reproductive organs that were evaluated. There were no microscopic findings in the testes or epididymides at 300 mg/kg/day, and no noteworthy changes in sperm parameters were observed.
Executive summary:

The objective of this study was to determine the potential toxicity of Cyclamen Aldehyde, when given orally via stomach tube for 14 consecutive days to male New Zealand White rabbits. In addition, potential effects on the male reproductive organs, including sperm parameters were assessed.

The study design was as follows:

Group No. No. of Male Rabbits Test Material Dose Level (mg/kg/day) Dose Concentration (mg/mL) Dose Volume (mL/kg)
1 5 Corn Oil 0 0 1
2 5 Cyclamen Aldehyde 30 300 1
3 5 Cyclamen Aldehyde 100 100 1
4 5 Cyclamen Aldehyde 300 300 1

Rabbits were administered the test substance, Cyclamen Aldehyde, and/or the control article, corn oil, formulations orally (stomach tube) once daily on Day 1 of study (DS 1) to 14.

The following parameters and endpoints were evaluated in this study: viability, clinical signs, body weights, body weight changes, food consumption, gross and microscopic pathology, sperm evaluations (motility, count, and morphology), and organ weights. Urine samples were collected from all rabbits on Day 15 prior to euthanasia for possible future evaluation.

All rabbits survived to scheduled euthanasia. None of the clinical signs that occurred were attributed to oral administration of Cyclamen Aldehyde, and all rabbits appeared normal at necropsy examination.

Body weights, body weight gains and food consumption values (g/day and g/kg/day) were comparable among the groups and were unaffected by dosages of Cyclamen Aldehyde as high as 300 mg/kg/day.

Cyclamen Aldehyde did not affect the weights of the reproductive (testes, epididymides, seminal vesicles [with and without fluid] or prostate) or non-reproductive (liver or kidneys) at any dosage level. There were no microscopic findings in the testes or epididymides at 300 mg/kg/day. In addition, there were no noteworthy changes in sperm motility (number and percentage of motile sperm or nonmotile sperm from ejaculated semen samples), sperm concentration (total and cauda epididymal sperm count and density) or sperm morphology at any dosage level.

In conclusion, Cyclamen Aldehyde when administered orally to male rabbits for 14 days at dosages as high as 300 mg/kg/day did not produce any clinical observations, changes in body weight or food consumption or affect the weights of any reproductive or non-reproductive organs that were evaluated. There were no microscopic findings in the testes or epididymides at 300 mg/kg/day, and no noteworthy changes in sperm parameters were observed.