3-p-cumenyl-2-methylpropionaldehyde

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Remarks:

An assessment of toxicokinetics, based on experimental and available data, in accordance with Annex VIII

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 Mar 2019 to 07 May 2019.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Species-specificity of metabolic fate and toxicity.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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

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.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

2020

Reference 3

Reference Type:

publication

Title:

Unnamed

Year:

2017

Materials and methods

Objective of study:

metabolism

other: Potential toxicity (mainly on reproductive organs) in male rats (Dose Range Finder)

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 Dose Range Finder 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 Cycla-men 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:

GLP facility: Charles River Laboratories Den Bosch B.V.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Details reported in Section 7.5.1; Key Study/Repeated dose toxicity 28-d: oral Range finder - CRL, 2020.

Sex:

male

Details on test animals or test system and environmental conditions:

Details reported in Section 7.5.1; Key Study/Repeated dose toxicity 28-d: oral Range finder - CRL, 2020.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Details reported in Section 7.5.1; Key Study/Repeated dose toxicity 28-d: oral Range finder - CRL, 2020.

Duration and frequency of treatment / exposure:

Details reported in Section 7.5.1; Key Study/Repeated dose toxicity 28-d: oral Range finder - CRL, 2020.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Dose Concentration: 6 mg/mL
Dose Volume: 5 mL/kg

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Dose Concentration: 20 mg/mL
Dose Volume: 5 mL/kg

Doses / concentrations 3

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 / concentration:

5

Positive control reference chemical:

no

Details on study design:

Details reported in Section 7.5.1; Key Study/Repeated dose toxicity 28-d: oral Range finder - CRL, 2020.

Details on dosing and sampling:

Details reported in Section 7.5.1; Key Study/Repeated dose toxicity 28-d: oral Range finder - CRL, 2020.

Statistics:

Details reported in Section 7.5.1; Key Study/Repeated dose toxicity 28-d: oral Range finder - CRL, 2020.

Results and discussion

Main ADME results

Type:

metabolism

Results:

4-isopropyl-benzoic acid (iPBA)

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Plasma and tissue samples from rats exposed orally to CA for 28 days showed a high circulating level of iPBA and its glucuronides which are the main circulating metabolites of CA. This would indicate absorption from the gut following first pass metabolism in the liver from the gut by both passive and active mechanisms. Permeation Absorption following respiratory exposure is expected to be limited based on the physical-chemical properties of the substance previously described. In the absence of specific absorption data via inhalation, the conservative default route-to-route extrapolation can be applied with pulmonary absorption set at 100% for risk assessment purposes. The physical-chemical properties of the substance indicate that dermal absorption would also be limited by a low rate of transfer between the stratum corneum and the epidermis. Nevertheless, based on the indicated sensitisation potential of the test item, some systemic uptake must occur although it may only be a small fraction of the applied dose. As a Tier I screen, in the absence of absorption data via this route, the ECHA (R8) default value of 50% adsorption is used for risk assessment.

Details on distribution in tissues:

Plasma and tissue samples from rats exposed to CA for 28 days showed a high circulating level of iPBA and its glucuron-icdes. Analysis of tissue samples indicated that a high amount of iPBA-CoA had accumulated in the liver and iPBA-CoA was also detected in the testes, albeit at much lower concentrations. Oxidation of CA to Cyclamen carboxylic acid and then to iPBA on the one hand, and hydroxylation of Cyclamen carboxylic acid are two key metabolic pathways. From iPBA, multiple secondary metabolites are formed, including conjugates to glucuronic acid, glycine, glutamate, carnithine and taurine. The acyl glucuronide and the glycine conjugate of iPBA were the most abundant phase II metabolites of CA detected in plasma. The acyl glucuronide of iPBA is, in addition to iPBA, an important metabolite in the testes and was also detected in liver samples.

Details on excretion:

It is expected that the various glucuronide conjugates previously described would be excreted into bile and urine. Glucuronide conjugates are extensively excreted via bile, and glucuronide conjugate metabolites of BMHCA, a read-across molecule to CA have been observed in urine of rats (Heike Laue et al, 2020. Benzoyl-CoA conjugate accumulation as an initiating event for male reprotoxic effects in the rat? Structure-activity analysis, species specificity and in vivo relevance, accepted for publication in Archives of Toxicology).

Metabolite characterisation studies

Metabolites identified:

yes

Remarks:

in vivo and in vitro studies identify 4-isopropyl-benzoic acid (iPBA) as the main metabolite

Details on metabolites:

Interspecies comparison of metabolism of Cyclamen aldehyde (dosed at 1, 10 and 100 μM) was compared in incubations with cryopreserved hepatocytes from mice, rats, rabbits and humans in suspension (0.3 × 106 viable cells / ml for mouse, 1 × 106 viable cells / ml for other species) (ECHA, 2016a). Incubations were conducted in duplicate with incubation times of 0, 1 and 4 h. The five main metabolite peaks observed were the direct oxidation product (Cyclamen carboxylic acid) and several glucuronide conjugates, i.e. a direct glucuronide of the aldehyde, the glucuronide of cyclamen alcohol as well as the glucuronide of a hydroxylated cyclamen alcohol. Cyclamen alcohol itself was not detected by LC-MS. These metabolites occurred at high levels in all four species. In rats, cyclamen carboxylic acid was also further degraded to 4-isopropyl-benzoic acid (iPBA). Levels of this metabolite was below detection limit in rabbit, human and mouse hepatocyte incubations, indicating a species difference in the metabolism to iPBA.
Cyclamen Aldehyde was further tested in plated primary hepatocytes from rats, rabbits and humans which were incubated for up to 22 h compared to the short term incubation in suspension hepatocytes. In plated rat hepatocytes, metabolism to iPBA is rapid and this intermediate is further conjugated to Coenzyme A (CoA). In rat hepatocytes, this CoA conjugate (iPBA-CoA) is rapidly formed and remains at constant levels for the entire duration of the experiment (22h). In rabbit and human cells, an initial formation of iPBA-CoA is also detected. However, the conjugate is cleared over time, and only low levels are detected after 22 h. Dose-dependent formation of iPBA-CoA was observed in rat hepatocytes exposed with different concentrations of CA (0-50 µM). Similar concentrations of the CoA conjugate (0.95 to 1.42 µM) were detected with 5, 10 and 50 µM CA. Significant difference between human and rat hepatocytes is observed. These data indicate that in rats, a sustained accumulation of iPBA-CoA conjugate is observed, which is not the case in hepatocytes from rabbits and humans.
In a 28 day range-finder study, analysis of plasma from the top dose group (300 mg/kg bw/d CA) showed a high circu-lating level of iPBA (264.6 ± 85.4 µM, combined free iPBA and its glucuronides). These levels of iPBAwere associated withmarked effects on sperm formation.. Analysis of the tissue samples indicated that iPBA-CoA had accumulated in the liver, reflecting in vivo the results obtained in the plated hepatocytes in vitro. In addition, iPBA-CoA was also detected in the testes, albeit at much lower concentrations. iPBA is further metabolised to multiple secondary metabolites, including conjugates to glucuronic acid, glycine, glutamate, carnithine and taurine. The acyl glucuronide and the glycine conjugate of iPBA were the most abundant phase II metabolites of CA detected in plasma. The acyl glucuronide of iPBA is, in addition to iPBA, an important metabolite in the testes samples exposed to the top dose and was also detected in liver samples. Both hydroxylation and side chain degradation in combination lead to hydroxylated iPBA which is also conjugated with glucuronide. Similar to the results in the suspension hepatocytes in vitro, the glucuronide of cyclamen alcohol is found in the plasma, but was interestingly not detected in the liver samples (Natsch et al,2020. A species specific metabolism leading to male rat reprotoxicity of Cyclamen Aldehyde. Paper submitted).

Any other information on results incl. tables

Table:4-iPBA and Cyclamen acid plasma concentration after 28 days gavage dosing

Dose Group	4 -iPBA (uM)	Cyclamen acid (uM)
1 (0 mg/kg/d)	<LOD	<LOD
2 (30 mg/kg/d)	<LOD	0.2 +/- 0.1
3 (100 mg/kg/d)	18.8 +/- 5.1	0.7 +/- 0.4
4 (300 mg/kg/d)	264.6 +/- 85.4	3.2 +/- 2.1

 

Applicant's summary and conclusion

Conclusions:

CA and its metabolites are widely distributed following oral exposure being detected in plasma, liver and male reproductive organs. In the rat, metabolites including the iPBA-CoA conjugate accumulate in the liver and are found in lower concentrations in the testes.CA is metabolized to para-substituted benzoic acid derivates and further transformed to Coenzyme A (CoA) conjugates. A number of para substituted benzoic acids (p-BA) and chemicals metabolized to p-BA have been found to have adverse effects on sperm viability, motility and morphology. These effects are putatively associated with the metabolism of p-BA to toxic intermediates. We have shown that p-BA lead to accumulation of high levels of p-alkyl-benzoyl-CoA conjugates in plated primary rat hepatocytes and most recently confirmed a very strong correlation between p-alkyl-benzoyl-CoA accumulation in rat hepatocytes and the toxic outcome. Species specificity was probed by comparing rat, rabbit and human hepatocytes, and p-benzoyl-CoA accumulation was found to be specific to the rat hepatocytes, not occurring in human hepatocytes. There was also very limited accumulation in hepatocytes from rabbits that are a non-responder species in in vivo studies. Tissues of rats treated with toxic doses of Cyclamen Aldehyde, were detected with p-isopropyl-benzoyl-CoA conjugates in the liver and in the testes indicating that the metabolism observed in vitro is relevant to the in vivo situation and the critical metabolite does also occur in the reproductive tissue. The effects observed in rats is correlated with high circulating level of iPBA (264.6 ± 85.4 µM, combined free iPBA and its glucuronides), in the liver, and also in the testes. These multiple lines of evidence further support benzoyl-CoA accumulation as a key initiating event for a specific group of male reproductive toxicants, and indicate a species specific effect in the rat that is not relevant for human health risk assessment.

Executive summary:

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 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.