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Link to relevant study record(s)

Reference
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:
key study
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.
Reason / purpose for cross-reference:
reference to same study
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.
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.
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.
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 / 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.
Type:
metabolism
Results:
4-isopropyl-benzoic acid (iPBA)
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).
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).

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

 

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.

Description of key information

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
50
Absorption rate - inhalation (%):
100

Additional information

In vitro studies (hepatocyte suspensions and plated hepatocytes) from multiple species (mouse, rat, rabbits and humans) and in vivo studies in rats have been conducted to examine the metabolism of CA (manuscript in preparation). Metabolites of CA are widely distributed in vivo being found in the plasma, liver and testes. In vitro, five major metabolites were detected - the direct oxidation product cyclamen carboxylic acid and several glucuronide conjugates including 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 mouse, rabbit and human hepatocyte incubations, indicating a species difference in the metabolism towards iPBA.

In plated primary hepatocytes from rats, metabolism to iPBA is fast and this intermediate is further conjugated to Coenzyme A (CoA). This CoA conjugate (iPBA-CoA) is rapidly formed and, remains at constant levels for the entire duration of the experiment (22h). In plated rabbit and human hepatocytes, an initial formation of iPBA-CoA is also detected. However, this metabolite is cleared over time, and only low levels are detected after 22 h. These data clearly indicate that in rats, a sustained accumulation of iPBA-CoA conjugate is observed, which is not the case in hepatocytes from rabbits and humans.

Plasma and tissue samples from rats exposed to CA for 28 days demonstrate metabolism data that is consistent with in vitro results. Plasma samples showed a high circulating level of iPBA (264.6 ± 85.4 uM, combined free iPBA and its glucuronides) and iPBA and its glucuronides are the main circulating metabolites of CA. Analysis of the 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. Further detailed analysis of the different samples with LC-HRMS allowed drawing a tentative in vivo metabolic map. 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.

It is expected that the various glucuronide conjugates 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).