Registration Dossier

Administrative data

Description of key information

Oral studies in different species:

28-day study (rats):

A 28 days range finder study was performed on Cyclamen Aldehyde (CA) prior to a 90 days sub-chronic toxicity study required by ECHA (ECHA, 2017). Animals were evaluated after necropsy for sperm parameters. Serum samples and tissue samples were preserved for additional analytical investigations. Male animals (n = 5 per group) were treated for 28 days by oral gavage with 0, 30, 100 and 300 mg/kg bw/d. No mortality, toxicologically relevant clinical signs nor any effects on food consumption were observed up to 300 mg/kg bw/d. At 100 mg/kg bw/d, adverse test item-related reduction of motile sperms, progressive sperm and number of cells with a normal morphology was noted . In addition, increased number of spermatocytes with a detached head/abnormal neck and decreased number of cells with a coiled tail were noted. However, these effects on spermatogenesis at the mid dose did not reach statistical significance and no gross lesions or correlating histopathological effects in the testis were noted. At 300 mg/kg/d, adverse lower body weight and body weight gain were observed starting in the first week of dosing. At histopathological examination of the testis, adverse degeneration of elongating spermatids and spermatid retention was observed in all males. Statistically highly significant reduction of the percentage of motile (-77.8%) and progressive sperms (-91%) and of sperms with normal morphology (-97%) was observed. Total sperm numbers were also significantly affected at this concentration (-39%) (Figure 1). Thus the LOAL for male reproductive toxicity was at 100 mg/kg bw/d based on the directional effects and 300 mg/kg bw/d for the statistically significant effects with treatment for 28 days.

90 -day Study (rats):

A 90-day subchronic toxicity study in rats was requested by ECHA upon review of the Cycalmen Aldehyde submission, despite the availability of a one-generation study with prolonged treatment. It was conducted under GLP according to the OECD guideline 408 (OECD, 2018) with four groups of each 10 animals per sex dosed daily by oral gavage with 0, 15, 30 and 120 mg/kg bw/d of CA.At the tope dose of 120 mg/kg bw/d, statistically highly significant reduction (p< 0.01) of the percentage of motile (-55%) and progressive sperms (-76%) and of sperms with normal morphology (-73%) was observed . Total sperm numbers were also reduced at this concentration (-29%), while no statistically significant effects were noted at the mid and low dose (15 and 30 mg/kg bw/d). These sperm effects correlated to lower weight of the epididymides and sperm granulomas and were considered to be adverse. Thus the LOAL for male reproductive effects was at 120 mg/kg bw/d upon this prolonged treatment duration. In the liver, hepatocellular hypertrophy was noted in males starting at 30 mg/kg/d and in females starting at 15 mg/kg/d, which correlated with higher liver weight. However, the hypertrophy was without any changes in liver enzyme activity or significant morphologic degenerative changes microscopically, and was therefore interpreted to be an adaptive effect, and non-adverse. Further mild effects were reduced body weight gain at top dose (8% lower for males and 5% lower in females), vacuolation of the urothelium in the urinary bladder and higher kidney and heart weights in females, that were not considered adverse. These latter findings were without microscopic or macroscopic correlate, generally low in magnitude and without any corroborative evidence of alteration of function, and hence considered to be not adverse. Thus, the NOAEL for general toxicity was at ≥ 120 mg/kg bw/d for males and females.

In summary, different studies observe similar effects of CA on male rat reproductive organs and function below the dose for general toxicity, while effects in females only occur at the dose of general toxicity. Depending on treatment duration, male reproductive function is affected with a NOAEL of 25 mg to 30 mg in the studies with treatment for 83 – 90 days while the NOAEL is at 100 – 125 mg / kg bw/d in the studies conducted for 14 – 28 days (100 mg /kg bw/d for directional effects). The NOAEL for general toxicity is occurring at the same or at one higher dose group, and is mostly based on changes in organ weights, especially on liver weights.

14 -day Study (Rabbits):

The potential toxicity of CA was studied by oral gavage dosing of 0, 30, 100 or 300 mg/kg bw/d for 14 consecutive days to groups of five male New Zealand White rabbits. Potential effects on the male reproductive organs, including sperm parameters were specifically assessed. Body weights, body weight gains and food consumption values were comparable among the groups and were unaffected by dosages of CA as high as 300 mg/kg/d. CA did not affect the weights of the reproductive (testes, epididymides, seminal vesicles [with and without fluid] or prostate) or non-reproductive (liver or kidneys) tissues at any dosage level. There were no microscopic findings in the testes or epididymides at the 300 mg/kg/d dose observed. In addition, there were no changes in sperm motility (number and percentage of motile sperm or non-motile sperm from ejaculated semen samples), sperm concentration (total and cauda epididymal sperm count and density) or sperm morphology at any dosage level. Thus, a NOAEL of ≥ 300 mg/kg bw/d for both general toxicity and for effects on male reproductive organs was determined for CA when administered orally to male rabbits for 14 days at dosages up to 300 mg/kg/d.

Cyclamen aldehyde (CA; 3-(4-isopropylphenyl)-2-methylpropanal) has been widely used for the last 100 years as a muguet note in perfumery. The safe use of this material is well established through the understanding of exposure and based on quantitative risk assessment confirmed by the RIFM Expert Panel, which is supported by a wide range of toxicology studies conducted over the last 20 years. Repeated dose studies in rats performed for hazard identification had indicated adverse effects on sperm maturation leading to impaired fertility. CA is structurally related to some other aryl substituted branched chain aromatic aldehydes, that have shown a propensity for spermatogenic effects in the rat. These aldehydes are metabolized by primary hepatocytes to para-substituted benzoic acid derivatives. An example of such an intermediary metabolite with reported male reprotoxicity in the rat is 4-tert-butyl-benzoic acid (p-tBBA). In rats, CA is efficiently transformed by oxidative degradation of the side-chain to 4-isopropyl-benzoic acid (iPBA)on the one hand and by hydroxylation (putative at the isopropyl group) on the other hand.

The effect on spermatogenesis appears to be linked to iPBA, the main circulating metabolite. iPBA and its conjugates were detected as dominant circulating metabolites, reaching a concentration of 264mM in plasma 24 h after the last dosing at the toxic dose of 300 mg/kg bw/d of CA. However, metabolism studies in rat, rabbit and human primary cultures of suspended hepatocytes, indicated species differences with iPBA readily formed by rat hepatocytes but below detection limit in cells from rabbits and humans. In plated rat hepatocytes, iPBA is detected as Coenzyme A-conjugate and this conjugate (iPBA-CoA) accumulates to rather stable levels over 22 h. It has been shown, that in vitro accumulation of CoA-conjugates is a metabolic hallmark strongly correlated to male rat reproductive toxicity for a number of structurally related compounds. iPBA-CoA is also formed in vivo both in the liver and in the testes of rats dosed with CA. iPBA-CoA does not accumulate in plated rabbit and human hepatocytes where it is rapidly cleared within 22 h. In a rabbit in vivo study, no effects of CA on spermatogenesis were observed. Thus, a species specific metabolic fate linked to CA toxicity in male rats can be postulated based on analytical data invitro and in vivo in the liver, and in male reproductive tissue in vivo. There is strong evidence that this species specific metabolic fate in the rat is not relevant to the rabbit, which is a non-responder species. Finally, lack of accumulation of iPBA-CoA in human hepatocytes indicates that like the rabbit, humans are unlikely to be vulnerable to iPBA hepatic and testicular toxicity.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
06 July 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment.
Reason / purpose for cross-reference:
reference to other study
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:
no
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
Species:
rabbit
Strain:
New Zealand White
Details on species / strain selection:
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.
Sex:
male
Details on test animals or test system 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.

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.
Route of administration:
oral: gavage
Details on route of administration:
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.
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.

The control article, corn oil, was dispensed once weekly for administration to Group 1 control rabbits. An adequate amount of the control article was dispensed into daily aliquots, which were stored at room temperature until used. The control article was stirred continuously during dosing. Details of the preparation and dispensing of the control article have been retained in the Study Records. Any residual volumes were discarded daily after the completion of dosage administration.

Test substance dosing formulations were prepared in accordance with Sponsor instructions at appropriate concentrations to meet dosage level requirements. The dosing formulations were prepared once weekly and dispensed into daily aliquots which were stored at room temperature, protected from light until used. The dosing formulations were stirred for at least 30 minutes before dosing and continuously during dosing. Details of the preparation and dispensing of the test substance have been retained in the Study Records. The bulk test substance was handled in a chemical fume hood. Any residual volumes were discarded daily after the completion of dosage administration
Justification of Route
The oral gavage route was selected for use to maximize systemic absorption.
Analytical verification of doses or concentrations:
no
Remarks:
Samples for concentration, homogeneity and/or stability were not collected or analyzed.
Duration of treatment / exposure:
14 consecutive days.
Frequency of treatment:
Once daily doses.
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Dose concentration (mg/ML) 30
Dose volume (mL/kg) 1
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Dose concentration (mg/ML) 100
Dose volume (mL/kg) 1
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Dose concentration (mg/ML) 300
Dose volume (mL/kg) 1
No. of animals per sex per dose:
5 males per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
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.
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.

Positive control:
No.
Observations and examinations performed and frequency:
In-life Procedures, Observations, and Measurements
Clinical observations, mortality, general appearance, body weight, food consumption.

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.
Clinical signs:
no effects observed
Description (incidence and severity):
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.
Mortality:
no mortality observed
Description (incidence):
All rabbits survived to scheduled euthanasia.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
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 and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Description (incidence and severity):
Urine samples were collected from all rabbits on Day 15 prior to euthanasia for possible future evaluation.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
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.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross lesions observed at necropsy examination. All rabbits appeared normal.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test substance-related microscopic changes were observed in the testes or epididymides examined.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
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.
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.
Key result
Dose descriptor:
NOEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed at highest concentration
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
stating that effects were observed, but considered negligible
Critical effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
other: no adverse effect observed

PROTOCOL 20010310: A 14-DAY STUDY OF CYCLAMEN ALDEHYDE BY ORAL (STOMACH TUBE) ADMINISTRATION IN MALE NEW ZEALAND WHITE RABBITS

 

TABLE 6 (PAGE 1): SUMMARY OF SPERM MOTILITY, COUNT AND DENSITY

 

GROUP

DOSE MATERIAL

DOSE LEVEL (MG/KG/DAY)a

 

1

CORNOIL 0

2

CYCLAMEN ALDEHYDE 30

3

CYCLAMEN ALDEHYDE 100

4

CYCLAMEN ALDEHYDE 300

RABBITS TESTED

N

5

5

5

5

SPERM MOTILITY

 

 

 

 

 

NUMBER MOTILE

 

MOTILE PERCENT

MEAN±S.D.

 

MEAN±S.D.

627.0

 

83.2

±

 

±

425.6

 

25.7

596.6

 

95.4

±

 

±

449.6

 

6.5

543.3

 

88.3

 

 

[

±   352.2

4]a

±   13.1

431.0

 

87.6

±

 

±

351.5

 

8.0

 

STATIC COUNT

 

 

 

 

 

 

 

 

[

4]a

 

 

 

(NONMOTILE)

 

TOTAL COUNT b

MEAN±S.D.

 

MEAN±S.D.

52.0

 

679.0

±

 

±

45.1

 

387.3

20.0

 

616.6

±

 

±

22.1

 

444.0

52.3

 

595.5

 

[

±   37.3

4]a

±   362.3

38.2

 

469.2

±

 

±

17.2

 

339.2

 

 

 

 

 

 

 

 

 

[

4]a

 

 

 

 

CAUDA EPIDIDYMAL SPERM COUNT

SPERM COUNT c

MEAN±S.D.

150.6

±

77.7

112.4

±

58.4

114.6

±   81.0

150.0

±

79.4

SPERM DENSITY d

MEAN±S.D.

434.56

±

165.83

353.00

±

124.28

262.64

±  177.50

389.90

±

189.66

a.  Excludes values for rabbit 8592, which 200 sperm were not located in twentyfields.

b.  Sum of number motile and static count. Groups of five fields were evaluated until a sperm count of at least 200 was achievedor

20 fields were evaluated.

c.  Sperm count used in the calculation of sperm density. Twenty fields wereevaluated.

d.  The sperm density was calculated by dividing the sperm count by the volume in the image area (76.6 x 10-6mL), multiplyingby

2 (dilution factor + 1) and multiplying by 10-6to obtain the sperm concentration. The calculated sperm concentration value (rounded to 1 decimal place) was multiplied by 50 (volume) and divided by the weight of the left caudaepididymis

(see Appendix 10, Table 2 for the weight of the left cauda epididymis) to obtain the sperm density. The calculated value will vary by approximately 0.8% from the Computer Automated Sperm Analysis because the digital image evaluated is slightlysmaller

(4 pixels) than the actual field causing a slight underestimate of the actual volume and an overestimate of theconcentration.

PROTOCOL 20010310: A 14-DAY STUDY OF CYCLAMEN ALDEHYDE BY ORAL (STOMACH TUBE) ADMINISTRATION IN MALE NEW ZEALAND WHITE RABBITS

 

TABLE 7 (PAGE 1): SUMMARY OF CAUDA EPIDIDYMAL SPERM MORPHOLOGY

 

------------------------------------------------------------------------------------------------------------------------------------ DAY 15 OF STUDY (DAY OF SACRIFICE)

------------------------------------------------------------------------------------------------------------------------------------GROUP                                      1                        2                        3                                                      4

DOSEMATERIAL                                    CORNOIL            CYCLAMENALDEHYDE        CYCLAMENALDEHYDE                                                 CYCLAMENALDEHYDE DOSELEVEL(MG/KG/DAY)a            0                                                           30                  100                      300

------------------------------------------------------------------------------------------------------------------------------------RABBITSEXAMINED     N                    5                        5                        5                                5

 

NORMAL                     MEAN±S.D.          141.6±17.2             121.4±30.5             102.2±25.6             128.2 ±31.3

 

PERCENTABNORMAL           MEAN±S.D.           30.4±9.5              31.1±9.1              48.9±12.8              35.9 ±15.7

 

DETACHEDHEAD              MEAN±S.D.           39.0±19.9              35.0±13.5              58.8±15.1              52.2 ±23.0

NOHEAD                    MEAN±S.D.           13.8±6.3              15.2±11.9              26.8±14.9              10.4 ±5.7

AMORPHOUS                  MEAN±S.D.            1.0±1.0               0.2±0.4               0.8±1.8               0.0 ±0.0

MACROHEAD                 MEAN±S.D.            0.0±0.0               0.0±0.0               0.2±0.4               0.2 ±0.4

MICROHEAD                 MEAN±S.D.            0.0±0.0               0.0±0.0               0.2±0.4               0.0 ±0.0

BROKENFLAGELLUM           MEAN±S.D.            4.0±2.3               2.2±1.1               2.4±1.7               3.8 ±4.8

COILEDFLAGELLUM           MEAN±S.D.            3.6±2.1               5.0±5.8               8.2±4.1               4.8 ±3.0

BENTFLAGELLUM             MEAN±S.D.            0.8±0.8               0.0±0.0               0.4±0.5               0.6 ±0.9

------------------------------------------------------------------------------------------------------------------------------------

a. Dosage occurred on days 1 through 14 of study.

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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1 Mar 2019 7 May 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Cyclamen aldehyde is oxidised to p-isopropyl-benzoic acid (iPBA) and further transformed to the coenzyme A conjugate 4-iPBA-CoA (p-iPBA-CoA). Coenzyme A conjugates are intracellular metabolites, which cannot be secreted and thus do not reach circulation, while the small acid 4-iPBA and conjugates of 4-iPBA with amino acid or glucuronide can potentially enter the bloodstream after being formed in the liver. Different chemicals acting as metabolic precursors of p-alkyl benzoic acid derivatives such as 4- iPBA have been found to affect spermatogenesis and reproductive capacity in male rats. These chemicals are efficiently transformed to p-alkyl-benzoyl Coenzyme A (CoA) conjugates in plated rat hepatocytes. A strong correlation was found between the reprotoxic potential and the ability of the chemicals to form p-alkyl-benzoyl CoA conjugates in liver cells [Laue, H., et al.,2017. However, so far most metabolic investigations were conducted in liver cells and not in cells from reproductive tissues and limited in vivo data are available.

The objectives of this study were:
a) Determine the potential toxicity of Cyclamen Aldehyde mainly reproductive organs of Male Wistar Han rats treated for 28 consecutive days by daily oral gavage at dose levels of 0, 30, 100 and 300 mg/kg/day;
b) Determine the circulating blood concentration of metabolites of Cyclamen Aldehyde in plasma sampled by GC-MS.
c) Determine the CoA-conjugate formation in tissue samples of both the testes and the liver after necropsy by LC-MS;
d) Determine the metabolite profile in tissue samples of both the testes and the liver and in plasma samples obtained on day 28 using high-resolution LC-MS analysis.

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, sperm analysis, gross necropsy findings and histopathologic examinations (testis only), plasma concentration of Cyclamen Aldehyde; CoA-conjugate formation in testes and liver, metabolite profile in the testes, liver and plasma samples obtained on day 28 of treatment.

GLP compliance:
no
Remarks:
Range finder
Specific details on test material used for the study:
Batch: PE00245593
Expiry date: 18 Jul 2020
Purity >= 98%
Records of the receipt, distribution, and storage of test item were maintained
Species:
rat
Strain:
Wistar
Remarks:
WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:
male
Details on test animals or test system and environmental conditions:
Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. The animals were 7 weeks old at initiation of dosing and weighed between 205 and 235 g. A health inspection was performed before the initiation of dosing.
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 6 days before the commencement of dosing.
On arrival and following randomization, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon type IV, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 21°C with an actual daily mean relative humidity of 39 to 54%.
Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
Water
Municipal tap water was freely available to each animal via water bottles.
Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Remarks:
Supplier: Sigma-Aldrich
Details on oral exposure:
Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly, filled out in daily portions and stored at room temperature. Formulations were stirred for at least 30 minutes before use. If practically possible, the dosing formulations and vehicle were continuously stirred during dosing. Adjustment was made for specific gravity of the vehicle and test item. Any residual volumes were discarded.

Sample Collection and Analysis
The Sponsor provided data that demonstrated that the test article was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data provided by the Sponsor have been retained in the study records.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Rats dosed for 28 days.
Frequency of treatment:
Once daily.
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Dose concentration: 6 mg/mL
Dose Volume: 5 mL/kg
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Dose concentration: 20 mg/mL
Dose Volume: 5 mL/kg
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Dose concentration: 60 mg/mL
Dose Volume: 5 mL/kg
No. of animals per sex per dose:
5
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist. The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).
Control animals:
yes, concurrent vehicle
Details on study design:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The total number of animals used in this study (5 males/group) was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Male Wistar Han rats, approximately 7 weeks of age on treatment Day 1 (for exact details see main study report), were administered Cyclamen Aldehyde extra via oral gavage daily for at least 28 consecutive days at dose levels of 30, 100 and 300 mg/kg bw/d.
Animals were assigned to groups by a stratified randomization scheme to achieve similar group mean body weights, with all animals within ± 20% of the sex mean. The dose levels were selected based on information provided by a14-day oral gavage study in rabbits.

Positive control:
No
Observations and examinations performed and frequency:
5 males/group

CAGE SIDE OBSERVATIONS: once daily throughout the Dosing Period. During the Dosing Period, these observations were performed after dosing. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Time schedule: twice daily (in the morning and at the end of the working day).

BODY WEIGHT: Weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

BIOANALYSIS:

Bioanalytical Sample Collection:
Prior to necropsy, blood was collected from the jugular vein. Blood samples at a target volume of 0.5 mL were collected into tubes containing K2-EDTA as anticoagulant. 4.9.1.2. Bioanalytical Sample Processing Samples were centrifuged within 2 hours after blood sampling at approximately 2000g for 10
minutes at 4-8 °C. Immediately after centrifugation, plasma was stored in labeled polypropylene tubes (Greiner Bio-One GmbH, Frickenhausen, Germany) at ≤ -75C until shipped on dry ice to the Sponsor. Samples were used to determine the circulating blood concentration of metabolites of Cyclamen aldehyde in
plasma sampled at the end of a 28 days range finder gavage study in rats by GC-MS. Collection on ice. Theoretical number of samples 21


Others: Testis, epididymis and liver were collected for metabolite analysis to determine the CoA-conjugate formation in tissue samples of both the testes and the liver, and to determine the metabolite profile in tissue samples of both the testes and the liver and in plasma samples obtained on day 28 using high-resolution LC-MS analysis.
Sacrifice and pathology:
Necropsy
All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

At necropsy, the left testis, right epididymis and liver were collected, stored in plastic bags and snap frozen into liquid nitrogen. Samples were stored at ≤ -75 C until shipment on dry ice to the Sponsor for metabolite analysis. The left epididymis was used for sperm analysis. The right testis was used for histopathology.

Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Tissue Collection and Preservation
Epididymis, Liver, Testis, Gross lesions/masses were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Histology
Tissues were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Histopathology
The right testis of each animal was examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. A peer review on the histopathology data was performed by a second pathologist.
Other examinations:
From all males, sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility was assessed from all samples and sperm smears were fixed for morphological evaluation. Abnormal forms of sperm from a differential count of 200 spermatozoa (if possible) per animal were recorded. Evaluation was performed for all males. One epididymis (left) from all males was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis were weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all males.

Plasma was collected to determine circulating blood concentration of metabolites of Cyclamen aldehyde at day 28. Testes, liver and plasma were collected to determine the CoA-conjugate formation. Metabolite profile were evaluated for plasma, testes and liver.
Statistics:
Body weight gain, food consumption were were summarized and statistically analyzed as indicated below according to sex and occasion.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Salivation was seen after dosing in all males at 100 and 300 mg/kg/day on most occasions. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect, its time of occurrence (i.e. after dosing) and as it was also seen in some control animals. This sign was considered to be a physiological response related to the taste of the test item rather than a sign of systemic toxicity.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related effects on body weight and body weight gain were observed in males at 30 mg/kg/day.

Slightly lower body weight and body weight gain was observed in males at 100 mg/kg/day starting on Day 15, with body weight being 0.93x of controls at the end of treatment (Day 28). Body weight and body weight gain in males at 300 mg/kg/day were moderately decreased starting on Day 8 (mean body weight was 0.89x of controls on Day 28), achieving statistical significance for body weight on Day 29.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No clear test item-related effects on food consumption were noted.
Food consumption was minimally lower at 100 and 300 mg/kg/day in Week 1, but lacked a dose-related effect and was therefore considered not to be toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related gross lesions were observed in the epididymis in males at 300 mg/kg/day.
A focal nodule (soft, yellow) was noted unilaterally in the tail of the epididymis in 3 out of 5 males at 300 mg/kg/day (No. 16 (right) and Nos. 18 and 19 (left)).
The remainder of the recorded macroscopic findings (prominent lobular architecture of the liver in one male (No. 17) and red foci in the kidney of one male (No. 18) at 300 mg/kg/day were within the range of background gross observations encountered in rats of this age and strain and were interpreted as likely to be unrelated to treatment with the test item.

Sperm Analysis:
No effects on sperm motility, concentration and morphology were observed in males at 30 mg/kg/day. The insufficient number of cells present for sperm cell morphology determination in one male at 30 mg/kg/day (No. 7) was considered to be an incidental finding and, in absence of any findings in the other 30 mg/kg/day animals, considered not to be toxicologically relevant.

At 100 mg/kg/day, a lower percentage of motile sperm (0.76x of control), progressive sperm (0.70x of control) and number of cells with a normal morphology (0.87x of control) was recorded. In addition, an increased number of cells with a detached head (4.43x of control) and abnormal neck (7.0x of control), and decreased number of cells with a coiled tail (0.39x of control) were observed. At 300 mg/kg/day, severe effects on the sperm motility, concentration and morphology were observed. These consisted of decreased total sperm count in the epididymis (0.61x of control), percentage of motile sperm (0.22x of control), percentage of progressive sperm (0.10x of control) and number of sperm cells with a normal morphology (0.04x of control).

The change in percentage of motile sperm and progressive sperm were statistically significant. The sperm cell morphology from 3 out of 5 males could not be determined as the sperm cell count for morphology was below the required 200 cells, which was considered to be caused by the test item. In addition, a lower number of cells with a coiled tail (0.13x of control), accompanied by an increase in number of cells with detached head (24.6x of control), abnormal head (2.0x of control) and/or neck (9.0x of control) and combined cells were observed in the remaining 2 out of 5 animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The right testis was evaluated histologically from all males. Test item-related microscopic findings were noted at 300 mg/kg/day group males and are summarized below. Reference to stages or steps in the description of the histologic changes refer to those described by Russel et al. (1990).

Testis Right (males treated with 300 mg/kg/d):
- Degeneration, elongated spermatids: Minimal (1); Moderate (4)
-Spermatid retention: Mild (1); Moderate (4)
-Degeneration, round spermatids: Moderate (1);
- Depletion, spermatid: Mild (1)

Degeneration of elongating spermatids was noted in all animals at 300 mg/kg/day, up to moderate degree. This was most readily observed in early tubular stages, approximately IVIII (corresponding to Step 15 to Step 19 spermatids), and was characterized by both an abnormal shape and abnormal location within the seminiferous tubules for the given stage. Abnormal shape was variable and consisted of either condensed, round, darkly basophilic nucleus with a bent/squiggled ‘neck’ giving a tadpole-like appearance to the nucleus, large cytoplasmic droplets extending into the lumen, and/or round shape with pale eosinophilic cytoplasm as a small condensed darkly basophilic nucleus which was often dissociated from the adjacent cells near the luminal border or sloughing into the lumen. Normally shaped elongated spermatids were not uncommonly present in the same tubular profile.

Spermatid retention was noted in all animals at 300 mg/kg/day, up to moderate degree. This was characterized by the presence of elongated spermatids (bothnormal and abnormal shaped) at the luminal surface of the seminiferous epithelium beyond the expected point of release (i.e. Stage VIII), and affected of primarily Stage IX-XII tubules. Less often, elongated spermatids were observed in low numbers at the base of the seminiferous tubules in all tubular stages.

Degeneration of round spermatids was prominent in one male at 300 mg/kg/day (No 18) and was characterized by condensed and hyper-eosinophilic round spermatids, often dissociated from the surrounding cells in the seminiferous tubule and sloughing into the lumen. Concurrent depletion of spermatocytes (round and elongated) and degeneration of elongated spermatids (as described above) were noted in this animal. The few remaining histologic changes noted in the testis, including minimal Sertoli cell vacuolation, were considered to be incidental findings and/or were within the range of background pathology encountered in the testis of rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

The combination of histologic changes noted in the testis of Cyclamen Aldehyde-treated male rats is suggestive of a test item-related abnormality in spermiogenesis (transformation of round spermatids to mature, elongated spermatids) and spermiation (release of mature spermatids from the seminiferous epithelium) (O’Donnell, 2014).
Increased vacuolation of Sertoli cells can be a test item-related change. However, in the present study vacuolation was observed in the controls as well and severity was minimal in all groups. Therefore, there was insufficient evidence to suggest a test item-related effect in the context of this study.
The changes noted by light microscopy correlate with the changes on sperm analysis including lower sperm concentrations, and morphologic abnormalities.
The nodules noted macroscopically in the epididymis of 3 of 5 males at 300 mg/kg/day are suggestive of sperm granulomas, however, the exact nature of these macroscopic changes requires histologic evaluation.

CONCLUSIONS
Adverse test item-related morphologic alterations following the administration of Cyclamen Aldehyde extra for 28 days to Wistar Han rats, were present in thetestis of males treated at 300 mg/kg/day. These adverse test item-related morphologic alterations consisted of microscopic spermatid degeneration, spermatid retention, and spermatid depletion in the testis, and macroscopic focal nodules in the tail of the epididymis.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Bioanalysis
Plasma:
Levels of Cyclamen aldehyde and Cyclamen alcohol were below detection limit in all plasma samples including the non-diluted plasma samples. 4-iPBA plasma concentrations were below detection limit in plasma samples collected from the control group and at 30 mg/kg/day. 4-iPBA was detected in all plasma samples at 100 and 300 mg/kg/day, ranging from 13.2 to 26.6 μM (100 mg/kg/day) and 151.1 to 385.0 μM (300 mg/kg/day). Average 4-iPBA concentrations were 18.8 ± 5.1 μM and 264.6 ± 85.4 μM at 100 and 300 mg/kg/day, respectively, corresponding to a 14-fold difference between the animals receiving the medium and the highest dose.

Cyclamen acid concentrations were below detection limit in plasma samples collected from the control group. Cyclamen acid was detected in all plasma samples of all test item groups and ranged from 0.1 to 0.3 μM (30 mg/kg/day), 0.3 to 1.2 μM (100 mg/kg/day) and 1.4 to 6.4 μM (300 mg/kg/day). Average Cyclamen acid concentrations were 0.2 ± 0.1 μM, 0.7 ± 0.4 μM and 3.2 ± 2.1 μM at 30, 100 and 300 mg/kg/day, respectively. Plasma concentrations of Cyclamen acid were 3.5-fold higher at 100 mg/kg/day compared to 30 mg/kg/day, whereas concentrations were 4.6-fold higher at 300 mg/kg/day compared to 100 mg/kg/day. However, plasma levels of Cyclamen acid were 27-fold lower (100 mg/kg/day) and 83-fold lower (300 mg/kg/day), respectively, compared to 4-iPBA.

Testes and liver:
In animals dosed with 30 mg/kg/day, trace amounts 4-iPBA-CoA were detected in the testes of only one individual. At 100 mg/kg/day, the conjugate was detectable at low levels in testes samples from all animals. At 300 mg/kg/day, 5-6 times higher levels (0.724 ± 0.222 nmol/gtissue) than at 100 mg/kg/day were observed, indicating that at the toxic dose this metabolite is significantly formed in the reproductive tissue. The concentration in the liver is clearly higher (> 500 fold), and strong accumulation in the liver of this metabolite as previously shown in the in vitro studies.

Metabolites
The major metabolite based on peak area observed by LC-MS analysis in the tissue samples and in the blood plasma is the acyl-glucuronide conjugate of iPBA (M25). The second most abundant peak is U1, an unknown metabolite with a mass of 208.1099 which was detected as the most abundant peak in plasma at 30 and 100 mg/kg/day and the second most abundant metabolite at 300 mg/kg/day. Further abundant metabolites were 4-iPBA (M3), hydroxylated 4-iPBA (M8), hydroxylated 4-iPBA-acylglucuronide (M37) and the glycine conjugate of 4-iPBA (M36).

Cyclamen aldehyde is easily oxidized to the corresponding acid (M2), but this is only a minor intermediate as shown before by GC-MS analysis of plasma samples and it was not detectable by LC-MS, neither in tissue nor in plasma samples. The acid is either directly degraded to iPBA or it is hydroxylated, putatively at the isopropyl-side chain. Hence the hydroxylated Cyclamen acid (M5) is a further important metabolite in this analysis especially in the plasma samples, where also the product of a further oxidation step is observed (di-acid, M6, detected in plasma mainly). M5 can be degraded to the hydroxylated iPBA (M8), which is found in tissue and plasma samples. However, M5 could be also formed from Cyclamen aldehyde by side chain degradation to 4-iPBA followed by hydroxylation. In both cases, the hydroxylated metabolites (M5 and M8) are then again conjugated, especially to glucuronic acid and esp. the glucuronide of M8 (M37) is quite abundant.

Next to the glucuronide, different iPBA conjugates are detected in the plasma and in testes and liver (glycine-, taurine-, carnitine- and glutamic acid-conjugates; M36, M28, M30 and M34). Thus in summary, the key metabolic pathways observed are formation of iPBA and subsequent conjugation mainly with glucuronic acid and glycine and/or hydroxylation and further oxidation.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other:
Remarks on result:
other: no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Sperm Analysis
Remarks on result:
other: male repro effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
In conclusion, administration of Cyclamen Aldehyde by once daily oral gavage for 28 days was well tolerated in male rats at 30 mg/kg/day. Test item-related lower body weight and body weight gain were observed at 100 and 300 mg/kg/day, which was considered to be adverse at 300 mg/kg/day.

Adverse test item-related morphologic alterations were present in the testis (microscopic) and epididymis (macroscopic) of males treated at 300 mg/kg/day and adverse test item-related changes in sperm motility, concentration and morphology were noted at 100 and 300 mg/kg/day.

The key metabolic pathways for Cyclamen Aldehyde observed are formation of iPBA and subsequent conjugation mainly with glucuronic acid and glycine and/or hydroxylation and further oxidation. 4-iPBA is efficiently conjugated to coenzyme A in the liver leading to high levels of this CoA conjugate. In the testes, iPBA-CoA was also found, but at substantially lower concentrations. Different iPBA conjugates are detected in the plasma and in testes and liver (glucuronide-, glycine-, taurine- carnitine- and glutamic acid-conjugates) with glucuronides as major Phase II metabolites.
Executive summary:

Cyclamen aldehyde is oxidised to p-isopropyl-benzoic acid (iPBA) and further transformed to the coenzyme A conjugate 4-iPBA-CoA (p-iPBA-CoA). Coenzyme A conjugates are intracellular metabolites, which cannot be secreted and thus do not reach circulation, while the small acid 4-iPBA and conjugates of 4-iPBA with amino acid or glucuronide can potentially enter the bloodstream after being formed in the liver. Different chemicals acting as metabolic precursors of p-alkyl benzoic acid derivatives such as 4- iPBA have been found to affect spermatogenesis and reproductive capacity in male rats. These chemicals are efficiently transformed to p-alkyl-benzoyl Coenzyme A (CoA) conjugates in plated rat hepatocytes. A strong correlation was found between the reprotoxic potential and the ability of the chemicals to form p-alkyl-benzoyl CoA conjugates in liver cells [Laue, H., et al.,2017. However, so far most metabolic investigations were conducted in liver cells and not in cells from reproductive tissues and limited in vivo data are available. Therefore, the objective of this study was to determine the toxicity on reproductive organs of male Wistar Han rats treated for 28 consecutive days by daily oral gavage at dose levels of 0, 30, 100 and 300 mg/kg/day. In the current study, clinical signs, body weights, food consumption, sperm analysis, gross necropsy findings and histopathologic examinations (testis only) were evaluated and correleted with

circulating blood concentration of metabolites of Cyclamen Aldehyde, CoA-conjugate formation in tissue samples (testes and the liver), as well as the metabolite profile in tissue samples of the testes and the liver.

Male Wistar Han rats were treated with Cyclamen Aldehyde for 28 consecutive days by daily oral gavage at dose levels of 0, 30, 100 and 300 mg/kg/day.

This current study indicates that 4-iPBA and the glucuronide conjugate of 4-iPBA are major circulating metabolites in the plasma of rats dosed for 28 days with cyclamen aldehyde especially at 300 mg/kg/day, where significant effects on sperm formation are observed. 4 - iPBA is efficiently conjugated to coenzyme A in the liver leading to high levels of this CoA conjugate. In the testes, the target organ for male reproductive toxicity, iPBA-CoA was also found, but at substantially lower concentrations. Different iPBA conjugates are detected in the plasma and in testes and liver (glucuronide-, glycine-, taurine- carnitine- and glutamic acid-conjugates) with glucuronides as major Phase II metabolites. Cyclamen aldehyde is easily oxidized to the corresponding acid (M2), but this is only a minor intermediate. It is either directly degraded to iPBA and then hydroxylated, putatively at the isopropyl-side chain (M8). Alternatively, the acid M2 can directly be hydroxylated to M5 which is a further important metabolite, and then degraded to the hydroxylated iPBA (M8) or further oxidized (M6). In both cases, the hydroxylated metabolites is conjugated, especially to glucuronic acid. Given that 4-iPBA is the putative toxic metabolite, this hydroxylation

pathway may be a competing detoxification pathway.

No mortality occurred in the study and there were no toxicological relevant clinical signs observed.

Slightly lower body weight and body weight gain were observed in males at 100 mg/kg/day. The body weight and body weight gain in males at 300 mg/kg/day were moderately decreased (achieving statistical significance for body weight on Day 29). At the severity observed, the body weight effects at 300 mg/kg/day were considered to be adverse, but the effects at 100 mg/kg/day were considered not to be adverse. Food consumption did not reveal any test item-related effects.

During sperm analysis, test item-related changes in motility, concentration and morphology were observed at 100 and 300 mg/kg/day. These consisted of a lower percentage of motile sperm and progressive sperm and number of cells with a normal morphology and coiled tail and an increase in number of cells with a detached head and abnormal neck at 100 mg/kg/day. At 300 mg/kg/day, the effects on sperm motility, concentration and morphology were similar compared to 100 mg/kg/day, but more severe. The changes in percentage of motile sperm and progressive sperm at 300 mg/kg/day were statistically significant. Additionally, decreased total sperm count in the epididymis and an increase in number of cells with abnormal heads and combined cells were observed at 300 mg/kg/day. In 3 out of 5 males at

300 mg/kg/day insufficient amount of cells were present to determine sperm cell morphology. The effects observed at sperm analysis at 100 and 300 mg/kg/day were considered to be adverse.

At necropsy, a focal nodule (soft, yellow) was noted unilaterally in the tail of the epididymis in 3 out of 5 males at 300 mg/kg/day. This finding is suggestive for sperm granulomas, however, the exact nature of this macroscopic change requires histological evaluation, which was not included in this study. Based on the type of finding and incidence, this finding was also considered test item-related and adverse.

At histopathological examination of the testis, degeneration of elongating spermatids and spermatid retention was observed in all males at 300 mg/kg/day. In addition, degeneration of round spermatids was prominent in one male at 300 mg/kg/day. The combination of histologic changes noted in the testis at 300 mg/kg/day is suggestive of a test item-related abnormality in spermiogenesis (transformation of round spermatids to mature, elongated spermatids) and spermiation (release of mature spermatids from the seminiferous epithelium) (O’Donnell, 2014). These changes correlated with the changes observed in the sperm

analysis, including lower sperm concentrations, and morphologic abnormalities.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
02 Jul 2019 - 9 Nov 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The OECD 408 was requested by ECHA decision CCH-D-2114361700-57-01/F. The study has been delayed due to various operational constraints. Please see the attached letter explaining the delays.

We are actually updating our IUCLID dossier with the new data from the OECD 408 study. In addition of OECD 408 data, a 28-day range finder study, was performed prior to the 90-day required by ECHA. Serum samples and tissue samples (testis and liver) were preserved to de-termine metabolite profile of Cyclamen Aldehyde, in order to understand the species specificity of the repro adverse effect observed in rats but not observed in rabbits treated at same doses. Also, to support the outcome, data from 2 papers under submission (Laue et al, 2020 submitted; Natsch et al, 2020 submitted) were referenced and discussed for the current submission (IUCLID dossier - Section 7.1 Toxicokinetics and section 7.5 Repeated Dose toxicity summary).
Consequently, we are expecting ECHA feedback regarding the Extended one-generation re-productive toxicity study (OECD 443) with respect to the OECD 408 results.

Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
June 2018
GLP compliance:
yes
Remarks:
OECD 408
Specific details on test material used for the study:
Batch: PE00245593
Expiry date: 18 Jul 2020
Purity >= 98%
Records of the receipt, distribution, and storage of test item were maintained
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han Rats
Details on species / strain selection:
The rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
Animals (rats) received on 03 Jul 2019, Crl: WI(Han) from Charles River Deutschland, Sulzfeld, Germany. The animals were 6-7 weeks old at initiation of dosing. Males weighed between 157 and 187 g and females weighed between 125 and 144 g.
- Housing:
On arrival and following randomization, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. The room(s) in which the animals were kept was documented in the study records.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water.

ENVIRONMENTAL CONDITIONS:
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 21°C with an actual daily mean relative humidity of 46 to 72%. The values that were outside the targeted range occurred for 11 out of 100 days with a maximum of 72% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.
12-hour light/12-hour dark cycle was maintained (except during designated procedures). Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

DETAILS OF FOOD AND WATER QUALITY:
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
Municipal tap water was freely available to each animal via water bottles.
During motor activity measurements, animals had no access to water for a maximum of 2 hours.
Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Remarks:
Corn oil, specific gravity 0.92
Details on oral exposure:
Preparation of Test Item:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily (test item was not pre-weighed in the formulation container). Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

Sample Collection and Analysis:
Dose formulation samples were collected for concentration analysis for all groups on Week 1 (days 1, 2 and 3), weeks 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13.
Dose formulation samples were collected for homogeneity analysis for groups 2 and 4 on Week 1 (days 1, 2 and 3), weeks 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.
All samples to be analyzed were transferred (at room temperature protected from light) to the analytical laboratory at the Test Facility.

Analytical Method: Analysis was performed using a validated analytical procedure (Test Facility Study No. 20185929).

Concentration Analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Homogeneity Analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.


Stability Analysis: Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20185929) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20185929.

Batch: PE00245593
Expiry date: 18 Jul 2020
Purity >= 98%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted weekly to assess accuracy and homogeneity.
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily (test item was not pre-weighed in the formulation container).
Concentration Analysis, Homogeneity Analysis and Stabiility Analysis were performed.
Analytical Method: Analysis was performed using a validated analytical procedure (Test Facility Study No. 20185929).

Concentration Analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Homogeneity Analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Stability Analysis: Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20185929) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20185929.


Duration of treatment / exposure:
A minimum of 90 days.
Frequency of treatment:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 90 days, up to and including the day before scheduled necropsy. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The first day of dosing was designated as Day 1.
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Dose concentration:: 7.5 mg/ML
Dose volume: 2 mL/kg
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Dose concentration:15 mg/ML
Dose volume: 2 mL/kg
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
Dose concentration: 60 mg/ML
Dose volume:2 mL/kg
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels of 15, 30 and 120 mg/kg/d were selected based on 28-day range finder oral gavage rat study to determine the potential toxicity of Cyclamen Aldehyde to male. Administration of Cyclamen Aldehyde by once daily oral gavage for 28 days was well tolerated in male rats at 30 mg/kg/day. Test item-related lower body weight and body weight gain were observed at 100 and 300 mg/kg/day, which was considered to be adverse at 300 mg/kg/day. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
Positive control:
There is no positive control.
Observations and examinations performed and frequency:
Mortality/Moribundity Checks: Throughout the study, animals were observed for general health/mortality and moribundity twice daily.
Detailed Clinical Observations: The animals were removed from the cage, and a detailed clinical observation was performed weekly, starting on Day 1 (prior to dosing), and on the day of necropsy.

Cage Side Observations: once daily, 0 to 1h after dosing.

Body Weights: Animals were weighed individually weekly, starting on Day 1. A fasted weight was recorded on the day of necropsy.

Food Consumption: quantitatively measured weekly starting on Day 1.

Water Consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Ophthalmic Examinations: The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/ml solution, THEA Pharma, Wetteren, Belgium) during Pretreatment in all animals, and at the end of the Dosing Period in Week 13 in all Group 1 and 4 animals.

Functional Tests: were performed on the first 5 animals per sex per group during Week 12-13. These tests were performed after completion of clinical observations.
 Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
 Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
 Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = hzabnormal/absent).
 Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
 Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

Estrous Stage Determination: Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage for evaluation of extreme thyroid hormone effects in females

Haematology:Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analyzed for the parameters specified in the following table. A blood smear was prepared from each hematology sample. Blood smears were labeled, stained, and stored. Blood smears were evaluated when required to confirm analyzer results.

Coagulation: Blood samples at a target volume of 0.45 mL were collected into tubes containing citrate as anticoagulant. Samples were processed for plasma, and plasma was analyzed for the parameters listed in the following table.

Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-heparin as anticoagulant. Samples were processed for plasma.
Sacrifice and pathology:
All animals surviving until scheduled euthanasia were weighed, and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy.

Necropsy: All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available .

Organ Weights: The organs identified in the following table were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratio (using the terminal body weight) were calculated. Organs Weighed at Necropsy: Brain,Cervix, Epididymisa, Gland, adrenal, Gland, pituitary, Gland, prostate,Gland, seminal vesiclea,Gland, thyroid, Heart, Kidneya, Liver, Ovary, Spleen, Testisa, Thymus, Uterus.

Tissue Collection and Preservation: Representative samples of the tissues identified in the following table were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Sperm Analysis: Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin for evaluation (see Appendix 1 for the deviation). One epididymis (left side) was removed, placed in labeled bags, and kept in the freezer at ≤ - 15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples. In the case of any abnormalities in the epididymis, the left side organ was fixed in modified Davidson's, and the right side organ was used for evaluation of sperm numbers. If abnormalities were found in epididymides (both sides), both these organs were fixed in modified Davidson's solution, and no evaluation of sperm numbers was performed.

Histology: Tissue Collection and Preservation: Artery, aorta, Body cavity, nasopharynx, Bone, femur, Bone marrow, Bone, sternum, Brain, Cervix, Epididymisa
Esophagus, Eyea, Gland, adrenal, Gland, clitoral, Gland, harderiana, Gland, lacrimal, Gland, mammary, Gland, parathyroid, Gland, pituitary, Gland, preputial,Gland, prostate, Gland, salivary,Gland, seminal vesicle, Gland, thyroid, Gross lesions/masses,Gut-associated lymphoid tissue, heart, Kidney, Large intestine, cecum Large intestine, colon, Large intestine, rectum, Larynx, Liver, Lung, Lymph node, mandibular, Lymph node, mesenteric, Muscle, skeletal, Nerve, optica, Nerve, sciatic, Ovary, Pancreas, Skin, Small intestine, duodenum, Small intestine, ileum, Small intestine, jejunum, Spinal cord, Spleen, Stomach,Testis,Thymus, Tongue, Trachea, Urinary bladder, Uterus, vagina, Vas deferensb (except animal identification, nasopharynx, bone marrow smears, clitoral gland, lacrimal gland, preputial gland, arynx, tongue and vas deferens) were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Histopathology: All tissues as defined above were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. A peer review on the histopathology data was performed by a second pathologist.
Statistics:
Any data collected during the predose period are tabulated, summarized or statistically analyzed. All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions were summarized in the main report and statistically analyzed as per the appropriate statistical method.

Variables analyzed:
Body Weight, Body Weight Gains, Food Consumption, Hematology Variables, Coagulation Variables, Clinical Chemistry Variables, Sperm Variables, Organ Weights, Organ Weight relative to Body Weight
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs were observed at 15 mg/kg/day.
Salivation seen after dosing in three females at 120 mg/kg/day between Days 17 and 55 and in a single male and female at 30 mg/kg/day was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Other clinical signs noted during the Dosing Period included fur loss, scabs and incidental erected fur, which occurred within the range of background findings to be expected for rats of this age and strain housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No effects on body weight and body weight gain were noted in males and females up to 30 mg/kg/day.

Slightly lower body weight gain was seen in males and females at 120 mg/kg/day starting at respectively Days 29 and 36 (statistically significant (p ≤ 0.05) in females). At the severity observed, these body weight effects were considered to be not adverse. Food consumption did not reveal any test item-related effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of treated animals was similar to the control level over the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmology findings that were related to treatment with the test item.

The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects on hematology parameters were observed.

A statistically significantly (p ≤ 0.01) lower platelets count was observed in females of all test item groups (up to 0.76x of controls). In absence of a dose-related response and based on the low magnitude of change, this finding was considered to be not test item related.

Other values in treated males and females achieving a level of statistical significance, when compared to controls, were considered to have arisen as a result of slightly high or low control values, occurred in the absence of a dose-related distribution and/or were, given the magnitude of change, considered to be of no toxicological significance.

Coagulation:
No test item-related effects on coagulation parameters were observed in females up to 120 mg/kg/day.

The statistically significant (p ≤ 0.01) changes in prothrombin time (PT) and activated partial thromboplastin (APTT) in treated males were, in absence of a dose-related response and based on the low magnitude of change, considered to be not test item related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related effects on clinical chemistry parameters were observed in males and females up to 30 mg/kg/day.

A statistically significantly lower total protein concentration was observed in males (p ≤ 0.05) and females (p ≤ 0.01) at 120 mg/kg/day (0.96x and 0.93x of controls respectively).

Furthermore, a statistically significantly (p ≤ 0.01) lower total cholesterol and HDL cholesterol was observed in males at 120 mg/kg/day (0.72x and 0.69x of controls, respectively).

Other values in treated males and females achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values, occurred in the absence of a dose-related distribution and/or were, given the magnitude of change, considered to be of no toxicological significance.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
The hind grip strength was statistically significantly (p ≤ 0.01) reduced in males at 120 mg/kg/day (0.69x of controls), but was still within the historical control range1.
Therefore, this finding was, in absence of any related clinical sign, considered to be not toxicologically relevant. Statistically significantly (p ≤ 0.01) higher total movements and ambulations were seen in females at 15 mg/kg/day (1.58x and 1.64x of controls respectively). With values remaining within historical control range2 and in the absence of a dose-related response, this finding was considered to be not test item-related. No effects were seen in the other test item groups.

All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No test item-related effects were seen in males at 15 mg/kg/day.

Test item-related organ weight differences were noted in the liver (males and females), kidney and heart (females), and the epididymis and seminal vesicle (males), as described below and summarized in Text table in section "Overall Information Results".

Higher liver weight was noted in males at 120 mg/kg/day, which was only statistically significant (p ≤ 0.01) relative to body weight. Higher liver weight was also noted in females starting at 15 mg/kg/day and was statistically significant (p ≤ 0.01) as absolute value and relative to body weight at all doses. Higher liver weights correlated with microscopic hepatocellular hypertrophy.

Higher kidney weight was noted in females only, starting at 30 mg/kg/day, and was statistically significant as absolute (p ≤ 0.05) value and relative to body weight (p ≤ 0.01) at 30 and 120 mg/kg/day. There was no microscopic correlate for this increased kidney weight.

In males, a statistically significantly (p ≤ 0.05) higher kidney weight was noted at 120 mg/kg/day only relative to body weight and was interpreted to be secondary to the difference in body weight.

A statistically significantly higher heart weight was noted in females only, starting at 30 mg/kg/day (p ≤ 0.01 at 30 mg/kg/day and p ≤ 0.05 at 120 mg/kg/day) when expressed relative to body weight. The differences in relative heart weight were slightly larger than proportional to the differences in body weight. The relevance of this finding is unclear, since was not followed by any microscopic changes.

A statistically significantly lower seminal vesicle gland weight was noted in males at 120 mg/kg/day (absolute value p ≤ 0.01 and relative to body weight p ≤ 0.05) and 30 mg/kg/day (relative to body weight only, p ≤ 0.05). There was no microscopic correlate. A statistically significantly (p ≤ 0.01) lower epididymis weight was noted in males at 120 mg/kg/day (absolute value only), which was without a microscopic correlate. There was no difference in the absolute testis weight. A statistically significantly (p ≤ 0.01) higher weight of the testis relative to body weight was noted in males at 120 mg/kg/day, which was secondary to the lower terminal body weights in that group.

There were no other test item-related organ weight changes. Any differences that reached statistical significance were either a reflection of the differences in terminal body weight, or were without a dose-relationship.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related macroscopic findings were noted in males up to 30 mg/kg/day and females up to 120 mg/kg/day.

A nodule was noted on the tail of the epididymis (unilateral, left) of one male treated at 120 mg/kg/day (No. 35). This correlated microscopically to a sperm granuloma.

The few remaining recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Sperm Analysis: No effects on sperm motility, concentration and morphology were observed in males up 30 mg/kg/day. At 120 mg/kg/day, statistically significantly (p ≤ 0.01) lower percentage of motile sperm (0.45x of control), progressive sperm (0.24x of control) and number of cells with a normal morphology (0.27x of control) was recorded. In addition, a statistically significant (p ≤ 0.01) increase in number of cells with a detached head (34.3x of control) and abnormal neck (23.0x of control) were observed. The sperm count in the epididymides was not statistically significantly reduced. A statistically significant (p ≤ 0.01) decrease in number of cells with a coiled tail was observed at 120 mg/kg/day (0.15x of control).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the liver, epididymis, and urinary bladder and are described below.

Hepatocellular liver hypertrophy up to mild degree was noted in males starting at 30 mg/kg/day, and in females starting at 15 mg/kg/day, up to mild degree, with a dose-related increase in incidence and severity. The hypertrophy was located predominantly in the centrilobular area, and occasionally extending to panlobular distribution (generally associated with mild severity grade). This finding correlated to higher liver weights.

Pigment in the liver was noted in females starting at 15 mg/kg/day, concurrent with hypertrophy, at minimal degree. Microscopically, the pigment was brown-green, located in the centrilobular region, and located in the sinusoidal lining cells. Single cell necrosis of hepatocytes was noted in one male at 120 mg/kg/day and one female at 30 mg/kg/day.

Vacuolation of the urothelium in the urinary bladder was noted in males and females at 120 mg/kg/day only, up to mild degree. This was characterized by multiple small, variably sized, well-delineated, round, clear vacuoles in the cytoplasm of umbrella cells.

Sperm granulomas in the epididymis (examined unilaterally) were noted in 3/10 males at 120 mg/kg/day only, at mild degree. In one male (Animal No. 35) this correlated to a nodule macroscopically.

Vacuolation of the pineal gland (when present in section) was noted in test item-treated males at a higher incidence and severity than that observed in the control males, however potential relationship of this finding to the test item is uncertain. The vacuolation was observed in 6 out of 8 males at 120 mg/kg/day (up to mild degree), 3 out of 8 males (up to moderate degree) and 2 out of 9 females (minimal) at 30 mg/kg/day, 5 out of 7 males at 150 mg/kg/day (up to mild degree) and 1 out of 4 control males (minimal). Due to the infrequent evaluation of this gland in routine toxicology studies, thus lack of historical control data and the difference in number of available glands for evaluation between groups, the assessment of this finding in relationship to the test item is difficult. Microscopically, pineal gland vacuolation was characterized by clusters of pinealocytes, with small to medium-sized, well-delineated, clear cytoplasmic vacuoles which did not cause notable increase in the size of the cells.

Remaining histologic changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormones
No test item-related effects on thyroid hormones were observed in males and females up to 30 mg/kg/day.

A decrease in triiodothyronine (T3) and thyroxine (T4) levels was seen in males at 120 mg/kg/day (0.69x and 0.45x of controls respectively), reaching statistical significance (p ≤ 0.01) for T4. T3 and T4 levels in females at 120 mg/kg/day were not statistically different from controls.

TSH remained within the historical control data range at these dose levels, and as such these changes were considered not to represent an adverse effect.

Other statistically significant changes in thyroid hormone parameters were at the low magnitude considered to be not toxicologically relevant.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
other: sperm analysis
Key result
Dose descriptor:
NOAEL
Effect level:
> 120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no test item effects in females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
cauda epididymis
seminal vesicle
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
no

Mean Percent Organ Weight Differences from Control Groups

 

 

Dose level (mg/kg/day)

 

15

Males 30

 

120

 

15

Females 30

 

120

BODY WEIGHT

-3.5

-0.8

-9.3*

0.4

-2.7

-7.1**

LIVER

 

 

 

 

 

 

Absolute

1.9

1.8

10.4

22.4**

23.1**

33.4**

Relative to body weight

5.5

2.4

21.4**

22.0**

26.5**

43.8**

KIDNEY

 

 

 

 

 

 

Absolute

0.5

0.4

-1.6

6.0

7.5*

8.2*

Relative to body weight

4.0

1.1

8.4*

5.7

10.5**

16.4**

HEART

 

 

 

 

 

 

Absolute

-3.9

-1.3

-6.0

5.1

9.7

4.0

Relative to body weight

-0.3

-0.4

3.6

4.9

12.8**

12.1*

EPIDIDYMIS

 

 

 

 

 

 

Absolute

-4.9

-6.7

-12.5**

 

 

 

Relative to body weight

-1.4

-5.7

-3.6

 

 

 

SEMINAL VESICLE

 

 

 

 

 

 

Absolute

-14.5

-19.6

-27.7**

 

 

 

Relative to body weight

-11.0

-18.6*

-20.1*

 

 

 

*: P<0.05, **: P<0.01;Bolded values were interpreted to be test item-related

Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals (Day 91-92)

 

 

Dose level (mg/kg/day)

 

0

 

15

Males

30

 

120

 

0

Females 15         30

 

120

LIVERa

Hypertrophy, hepatocellular Minimal

Mild

PigmentMinimal

Single cell necrosis Minimal

 

URINARY BLADDERa

Vacuolation, urothelial Minimal

Mild

 

EPIDIDYMISab

Sperm granuloma Mild

10

10

10

10

10

10

10

10

3

3

5

8

6

5

4

1

3

4

1

1

10

10

10

10

10

10

10

10

4

7

1

1

10

10

10

10

 

3

a  = Number of tissues examined from each group; b = unilateral examination

Conclusions:
Administration of Cyclamen Aldehyde by once daily oral gavage for at least 90 days was well tolerated in male and female Wistar Han rats up to 30 mg/kg/day. Adverse test item-related morphologic alterations were present in the epididymis (lower weight and granulomas) and in sperm (including alterations in motility, concentration and morphology) in males at 120 mg/kg/day. No adverse findings were observed in any of the females.

Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 30 mg/kg/day for males and at least 120 mg/kg/day for females.
Executive summary:

Wistar Han rats were treated with Cyclamen Aldehyde for at least 90 days by daily oral gavage at dose levels of 0, 15, 30 and 120 mg/kg/day.

Test formulations prepared were considered homogeneous at the concentrations tested. Accuracy results were only slightly below the acceptable range on a few occasions, but the majority of the analyses were within target agreement. Therefore, it was concluded that the formulations were prepared accurately.

The only observed test item-related clinical sign was salivation, which was observed directly after dosing in few animals at 30 and/or 120 mg/kg/day. This finding was considered to a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Slightly lower body weight gain was seen in males and females at 120 mg/kg/day starting at respectively Days 29 and 36 (statistically significant (p ≤ 0.05) in females). At the severity observed, these body weight effects were considered to be not adverse. Food consumption did not reveal any test item-related effects.

At clinical chemistry, a statistically significantly lower total protein concentration in males (p ≤ 0.05) and females (p ≤ 0.01) and statistically significantly (p ≤ 0.01) lower cholesterol and HDL cholesterol in males at 120 mg/kg/day were observed. These minor changes were in absence of a histopathological correlation and at the low magnitude of change, considered to be not adverse due to its severity.

A decrease in triiodothyronine (T3) and thyroxine (T4) levels in males at 120 mg/kg/day was observed, reaching statistical significance (p ≤ 0.01) for T4. A possible adversity of these changes could not be established within this study. However, there were no changes in TSH or any gross and microscopic findings in the thyroid gland.

During sperm analysis, test item-related changes in motility, concentration and morphology were observed at 120 mg/kg/day. These consisted of a statistically significantly (p ≤ 0.01) lower percentage of motile and progressive sperm and number of cells with a normal morphology, along with a lower total sperm count in the epididymides (not statistically significant). Additionally, a marked increase (statistically significant (p ≤ 0.01)) in number of cells with a detached head and abnormal neck were recorded. These sperm effects correlated with observed lower weight and sperm granulomas in the epididymides of these males. A

correlate to lower weight was not detected on microscopic examination. Microscopic sperm granulomas were noted in three out of ten males (one with correlating macroscopically

nodule), which was considered to be higher than expected to occur spontaneously, and may correlate indirectly to the changes noted in this group on sperm parameters. At the magnitude of change, the findings in sperm analysis and epididymis were considered to be adverse. Furthermore, a statistically significant (p ≤ 0.01) decrease in number of cells with a coiled tail was observed at 120 mg/kg/day, which was considered to be caused by the high incidence of the other sperm morphology alterations and was not a direct test item effect. No histologicchanges were noted in the testes.

Further test item-related microscopic findings were noted in the liver and urinary bladder.

In the liver, hepatocellular hypertrophy was noted in males starting at 30 mg/kg/day and in females starting at 15 mg/kg/day, which correlated with statistically significantly (p ≤ 0.01) higher liver weight. The hypertrophy was without any changes in liver enzyme activity or significant morphologic degenerative changes microscopically, and was therefore interpreted to be non-adverse. Furthermore, pigment in the liver was noted in females starting at 15 mg/kg/day, concurrent with hypertrophy. Microscopically, the pigment was brown-green and located in the centrilobular region and sinusoidal lining cells. The nature of the pigment cannot be definitively determined on routine H&E staining, but the brown-green color is suggestive of bilirubin and/or hemosiderin. No changes in total bilirubin were noted in clinical chemistry results, and no degenerative changes noted microscopically, thus the presence of minimal pigment was considered non-adverse. One male and one female at 120 mg/kg/day showed single cell necrosis of hepatocytes histologically, but at this low incidence and severity and without any notable concurrent individual clinical chemistry changes this was interpreted as non-adverse.

In the urinary bladder, vacuolation of the urothelium was observed in males and females at 120 mg/kg/day, which was considered to be not adverse, as there was no degeneration or necrosis associated with the minimal or mild cytoplasmic vacuolation observed. Vacuolationof the urinary bladder epithelium is considered as a nonspecific lesion.

Other test item-related findings included statistically significantly higher kidney (p ≤ 0.05) and heart weight (p ≤ 0.01 at 30 mg/kg/day and p ≤ 0.05 at 120 mg/kg/day) in females starting at 30 mg/kg/day. These findings were without microscopic or macroscopic correlate, generally low in magnitude and without any corroborative evidence of alteration of function, and were therefore considered to be not adverse. In addition, a statistically significantly (p ≤ 0.01) lower seminal vesicle gland weight was noted in males at 120 mg/kg/day (absolute value p ≤ 0.01 and relative to body weight p ≤ 0.05) and 30 mg/kg/day (relative to body weight only, p ≤ 0.05). The cause of this finding remains unclear, but may be influenced by lower body weights. In absence of a histopathological correlation and as these results remained within historical control range3, this finding was considered to be not adverse. Pineal gland vacuolation in the brain was observed at a higher incidence in males compared to controls. Considering the results from all dose levels (including 15 and 30 mg/kg/day), the incidence and severity (up to moderate) of pineal gland vacuolation was higher in males treated with the test item compared to control males, but without a dose-relationship. In females, pineal gland vacuolation was only observed in two out of ten females at 30 mg/kg/day. As no degenerative or necrotic changes were noted in association with the vacuolation, the relationship of the pineal gland vacuolation with the test item remains unclear, but is considered to be not adverse.

No mortality occurred and no test item-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, ophthalmoscopy, food consumption, hematology and coagulation).

Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 30 mg/kg/day for males and at least 120 mg/kg/day for females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
13
Species:
rat
System:
male reproductive system
Organ:
cauda epididymis
seminal vesicle

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

Mode of Action Analysis / Human Relevance Framework

Cyclamen aldehyde (CA) has been widely used for the last 100 years as a muguet note in perfumery. The safe use of this material is well established through the understanding of exposure and based on quantitative risk assessment confirmed by the RIFM Expert Panel, which is supported by a wide range of toxicology studies conducted over the last 20 years. Repeated dose studies in rats that were mainly conducted for the purposes of hazard identification for the REACH registration, revealed adverse effects on sperm maturation leading to impaired fertility. The effect on rats spermatogenesis appears to be linked to the main circulating metabolite, 4-isopropyl-benzoic acid (iPBA). However, metabolism studies in rat, rabbit and human primary cultures of suspended hepatocytes, indicated species differences with iPBA readily formed by rat hepatocytes but below detection limit in cells from rabbits and humans. In plated rat hepatocytes, iPBA is detected as Coenzyme A-conjugate and this conjugate (iPBA-CoA) accumulates to stable levels over 22 h. It has been shown, that in vitro accumulation of CoA-conjugates is a metabolic hallmark strongly correlated to male rat reproductive toxicity for a number of structurally related compounds. iPBA-CoA is also formed in vivo both in the liver and in the testes of rats dosed with CA. iPBA-CoA does not accumulate in plated rabbit and human hepatocytes where it is rapidly cleared within 22 h. In a rabbit in vivo study, no effects of CA on spermatogenesis were observed. Thus, a species specific metabolic fate linked to CA toxicity in male rats can be postulated based on analytical data in vitro and in vivo in the liver, and in male reproductive tissue in vivo. There is strong evidence that this species specific metabolic fate in the rat is not relevant to the rabbit, which is a non-responder species. Finally, lack of accumulation of iPBA-CoA in human hepatocytes indicates that like the rabbit, humans are unlikely to be vulnerable to iPBA hepatic and testicular toxicity.

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The dermal and inhalatory routes were waived on scientific grounds due to the justifications listed below. As such the oral route has been reported, and the data is available as requested by ECHA..

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In line with Column 2, point 8.6.1, Annex VIII of Regulation 1907/2006, a repeat-dose inhalation study does not need to be performed as the substance has low vapour pressure and high melting point, so the potential for the generation of inhalable forms is low. As an objective of Regulation EC No. 1907/2006 is to reduce, replace or refine animal testing, based on the above information and information in this dossier, it can be reasonably expected that inhalation exposure is not expected and as such, it is not warranted to test the hypothesis in animals.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
In line with Column 2, point 8.6.1, Annex VIII of Regulation 1907/2006, a repeat-dose inhalation study does not need to be performed as the substance has low vapour pressure and high melting point, so the potential for the generation of inhalable forms is low. As an objective of Regulation EC No. 1907/2006 is to reduce, replace or refine animal testing, based on the above information and information in this dossier, it can be reasonably expected that inhalation exposure is not expected and as such, it is not warranted to test the hypothesis in animals.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The physicochemical and toxicological properties suggest low potential for significant rate of absorption through the skin. Furthermore the results of laboratory animal studies show low acute dermal toxicity.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
The physicochemical and toxicological properties suggest low potential for significant rate of absorption through the skin. Furthermore the results of laboratory animal studies show low acute dermal toxicity.

Justification for classification or non-classification

On the basis of observations from appropriate reported studies, there is strong evidence that this species specific metabolic fate in the rat is not relevant to the rabbit, which is a non-responder species, and humans, given the lack of accumulation of the toxic metabolite (iPBA-CoA) in human hepatocytes. Like the rabbits, humans are unlikely to be vulnerable to iPBA hepatic and testicular toxicity.