4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 February 1995 - 15 August 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

other: EPA Pesticide Assessment Giudelines, Subdivision F, 40 CFR Part 158, 85-1

Version / remarks:

October 1982

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Remarks:

BOR:WISW

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann Versuchstierzucht GmbH & Co KG, Borchen, Germany
- Age at study initiation: approximately 8 weeks (males), approximately 11 weeks (females)
- Weight at study initiation: about 200 g
- Housing: 5 animals in Makrolon type III cages (acclimatization period), individually in Makrolon metabolism cages (during study)
- Diet: Altromin 1324 standard feed for rats, 18 g per animal/day
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 -25
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: acetonitile and 0.5% aqueous tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The radiolabelled compound was dissolved in acetonitrile and stored at approximately 4°C. The non labelled test compound was stored in solid form. For preparation of each dose the labelled test compound in acetonitrile was pipetted and blown dry with a gentle stream of nitrogen prior to adding 0.5% aqueous tragacanth suspension. Preparation of a homogenous suspension required agitation of the test substance with the tragacanth suspension at 50°C to 70°C for at least 15 min using supersonics and/or stirring at the same temperature for approximately 16 h. If needed non-labelled parent compound was weighed, dissolved in acetonitrile and mixed with the radioactive test solution prior to evaporation with nitrogen.

Duration and frequency of treatment / exposure:

single administration

Dose / conc.:

1.5 mg/kg bw/day (actual dose received)

Remarks:

males and females

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

males

No. of animals per sex per dose / concentration:

5 (low dose, both males and females)
5 (high dose, males)

Control animals:

no

Details on study design:

- Dose selection rationale: considering the low toxicity and the limited solubility of the compound the low dose was set at 1.5 mg/kg bw/day and the high at 75 mg/kg bw/day

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: expired air, urine, cage washes, faeces, blood, plasma, serum, organs and tissue (erythrocytes, spleen, gastrointestinal tract, bone, lung and muscle, adrenal glands, thyroid, ovaries, renal fat and uterus).
- Time and frequency of sampling: 0-4, 4-8, 8-24, 24-48 and 48-72 h (for tests 1 and 2) postadministration (urine), 0-24, 24-48 and for tests 1 and 2 also 48-72 h after dosing (faeces), collected 0.08, 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 24, 32, 48 h and for test 1 also 72 h after administration (blood), 0-8, 8-24, 24-48 and 48-72 h after dosage test 1(expired air), at necropsy 72 h postadministration (test 1,2) and 48 h postadministration (test 3,4), organs and tissues were homogenized for analysis.
- Other: in tests 1 and 2 animals were sacrificed 72 h postadministration, test 3 and 4 were shortened to 48 h thereafter, because of a rapid excretion of radioactivity in the first tests.
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, cage washes, faeces.
- Time and frequency of sampling: 0-48 h collection period and 0-24 h collection period (urine), 0-24 h collection period (faeces).
- From how many animals: 0-48 h collection period, urine of rats of test 1 anf 3 were combined for analysis of unknown metabolites, aliquots of urine of the collection periods 0 - 48 h of the rats of tests no. 1, 2, 3, and 4 (0 -24 h) were combined to separate samples per dose group and sex (urine), faeces of cellection period 0 - 24 h were combined per dose group and sex of tests 1 to 4 (faeces).
- Method type(s) for identification: HPLC-MS-MS, TLC, NMR-Spectroscopy.
- Limits of detection and quantification: a radioactive peak was regarded as relevant having a signal to noise ratio of at least 2.5.

Preliminary studies:

In a preliminary test the expiration of CO2 and other labelled volatiles was followed over a period of 72 h using male rats (test 1). In this test < 0.05% of the administered dose were expired within 72 h.

Type:

absorption

Results:

The test substance was absorbed rapidly from the gastrointestinal tract of rats after oral administration. In the high dose group absorption was at least 44% in males and >80% in low dose male and female rats.

Type:

distribution

Results:

< 5% of the administered dose remained in the body in males and 7% and more in females, liver followed by kidneys, lungs and thyroid gland contained significant amounts.

Type:

metabolism

Results:

The test substance was metabolized extensively. Two major groups of metabolites (the hydrolysis product containing the radiolabelled moiety either as free Cyclohexyl ethylamine (CEA) and Cyclohexylamine (CA) or as hydroxylated CEA) were identified.

Type:

excretion

Results:

Total excretion was rapid. >92% was excreted within 48 h or 72 h at both doses in urine and faeces. The ratio between renal to faecal excretion was 4:1 to 3:1 in males and females.

Details on absorption:

Absorption of the radio-labelled test substance started immediately after dosing and was absorbed rapidly from the gastrointestinal tract of the rats in all dose groups after oral administration of a suspension in 0.5% aqueous tragacanth. The absorption of the test substance amounted to at least 44% (high dose male animals) and >80% (low dose male and female animals), given as the renal portion plus the sum of the organs without gastro-intestinal tract. The maximum equivalent concentration was reached about 70 to 100 min postadministration in the low dose groups and at 180 min in the high dose males. The dose normalized equivalent concentration peaked at P=0.14 for the high dose group and at P=0.78 and 0.54 for the low dose groups and thus between 14% and 78% of the equidistribution concentration when comparing the average values for five animals in each test. The plasma maximum was independent of the sex and not proportional to the administered dose, the latter indicating incomplete absorption and unchanged parent test compound in the faeces of the high dose animals.

Details on distribution in tissues:

At both doses a moderately fast decrease of redioactivity concentration in plasma, due to fast distribution and excretion was observed. Elimination half-lives were relatively short and ranged from about 16 to 37 h. The total clearance was relatively high in all dose groups, 1.1 to 7.5 [mL/(min*kg bw)] without correction for the bioavailability. The renal clearance (CLR = CL x % renal excretion) was moderately high, 0.7 and 7.5 [mL/(min*kg bw)]. These numbers are in the range of the maximum glomerular filtration capacity of the kidney meaning that the bulk of radioactivity was eliminated via glomerular filtration without reabsorption. It was shown that the different dose levels seem to influence the distribution behaviour of total radioactivity in the body. The mean residence time MRT of the total radioactive dose in the plasma was short in all dose groups, about 24 and 43 hours, indicating that elimination is a moderately fast process. The radioactivity remaining in the body without the gastrointestinal tract was 4.5% to 4.0% of the dose at sacrifice in low dosed males (tests 1 and 2), 6.7% for low dosed females (test 3) and 2.8% for high dosed males. Significant amounts of radioactive residues were found in the liver and to a minor extent in the kidney. The residue in males was 0.7 µg/g and in females 1.2 µg/g (low dose group) and 7.2 µg/g in high dosed males. Among the organs and tissues checked liver in the low dose group was found to contain the highest dose normalized equivalent concentration of P = 0.45 for the males and P = 0.89 for the females, i.e. 45% and 89% of the equidistribution concentration whereas it amounted to 0.17 for the (high dose males, thus indicating a much lower absorption at the high dose level. Kidney exhibited between 21% and 32% of the radioactivity in the liver. In the low dose groups also the lungs and the thyroid gland showed relatively high residues.

Details on excretion:

After oral administration on average 92.7% of the recovered radioactivity was excreted via urine and faeces within 48 h of the female animals and 95.4% or more within 72 h of the low dose males and 97.0% of the high dose males after 48 h. The majour route of excretion was renal (ratio renal to faecal in in low dose groups: 3.9:1 to 5.5:1 and in high dose groups 0.7:1). The excretion pattern of total radioactivity was not significantly different between male and female rats. Only slightly different retention behaviour of radioactive residues was observed between male and female rats: females retained almost twice the amount as compared to males.

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 16.1 h

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 37.0 h

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 16.4 h

Test no.:

#2

Toxicokinetic parameters:

AUC: 24.6

Remarks:

µg/(gxh)

Test no.:

#3

Toxicokinetic parameters:

AUC: 25.9

Remarks:

µg/(gxh)

Test no.:

#4

Toxicokinetic parameters:

AUC: 166

Remarks:

µg/(gxh)

Test no.:

#2

Toxicokinetic parameters:

Cmax: 1.01

Remarks:

µg/g

Test no.:

#3

Toxicokinetic parameters:

Cmax: 1.71

Remarks:

µg/g

Test no.:

#4

Toxicokinetic parameters:

Cmax: 6.38

Remarks:

µg/g

Metabolites identified:

yes

Details on metabolites:

In the low dose group unchanged test substance was only detected at minor acounts in faeces. In high dosed males unchanged test substance was observed as major component in faeces extracts. In the urine no parent compound was detectable. Main metabolites identified in urine were cyclohexyl-ethylamine (CEA) and cyclohexylamine (CA). These free amines accounted for 7.5% and 4.2% (CEA, CA, test 4), 18.3% and 13.4% (test 3) and 9.7% to 12.4% and 10.7 to 11.7% (test 2, test 1) in the sum of urine and faeces extracts, females showing a slightly different pattern with less hydroxylated and more unchanged CEA and CA. In addition six minor metabolites (0.2% to 4.1%) were identified in urine. They belong to two groups, one baring the CEA-carbamoyl moiety linked to amino groups of endogenous acids (cyclohexyl ethyl carbamoyl-tauric acid [CEA-CO-tauric], cyclohexyl ethyl carbamoyl glutamic acid [CEA-CO-glutamic] , hydroxy-cyclorjexyl ethyl carbamoyl glutamic acid [HO-CEA-COglutamic] and cyclohexyl ethyl carbamoyl hydroxyglutamic acid [CEA-CO-HO-glutamic]) and the other one baring the CA-carbamoyl group linked to the nitrogen of ammonia (cyclohexylurea), urea (cyclohexylbiuret) and the sulfur of mercapturic acid (cyclohexyl carbamoyl mercapturic acid [CA-CO-S-cys-N-ac]). No sulfate nor glucuronic acid conjugates were found. In males of the high dose group a proportionally lower amount of metabolites were found in faeces, but apprimately 19% of unchanges parent compound was detected. The total rate of identification was relatively high in the low dose group (68% to 78%) of the administered dose (73% to 75% of the total recovered). The identification was lower in the high dose group (63% of the dose and 64% of the recovered). In the low dose tests the sum of the main metabolites hydroxy-cyclohexyl-ethylamine (HO-CEA), CEA and CA amounted to 62% to 65% of the recovered radioactivity, females showing more of the unhydroxylated amines than males. In the high dose test the unchanged substance was a major compound and was about 61% together with the three amines. The other metabolites found preferentially in urine were minor to trace metabolites with lower relative amounts in the high dose test.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October 1994 - 03 March 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

other: volatility

Qualifier:

according to guideline

Guideline:

other: EPA Pesticide Assessment Giudelines, Subdivision F, 40 CFR Part 158, 85-1

Version / remarks:

October 1982

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Details on species / strain selection:

BOR:WISW (SPF Cpb) later changed to Hsd/Win:WU and Hsd Cpb:WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann Versuchstierzucht GmbH & Co KG, Borchen, Germany
- Age at study initiation: approximately 8 weeks (males), approximately 11 weeks (females)
- Weight at study initiation: about 200 g
- Housing: 5 animals in Makrolon type III cages (acclimatization period), individually in Makrolon metabolism cages (during study)
- Diet: Altromin 1324 standard feed for rats, 18 g per animal/day
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 25
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: oral: gavage and intraduodenal

Vehicle:

other: acetonitrile and 0.5% aqueous tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The radiolabelled compound was dissolved in acetonitrile and stored at approximately 4°C. The non labelled test compound was stored in solid form. For preparation of each dose the labelled test compound in acetonitrile was pipetted and blown dry with a gentle stream of nitrogen prior to adding 0.5% aqueous tragacanth suspension. Preparation of a homogenous suspension required agitation of the test substance with the tragacanth suspension at 50°C to 70°C for at least 15 min using supersonics and/or stirring at the same temperature for approximately 16 h. If needed non-labelled parent compound was weighed, dissolved in acetonitrile and mixed with the radioactive test solution prior to evaporation with nitrogen.

Duration and frequency of treatment / exposure:

1. single oral dose (high and low dose)
2. repeated oral dose 15 x 1.5 mg/kg bw/day (non-radioactive compound once per day and 24 h after the 14th non-radioactive dose a single radioactive dose)
3. single intraduodenal dose
Please refer to table 1 in the "Any other information on materials and methods incl. tables" section.

Dose / conc.:

1.5 mg/kg bw/day

Remarks:

1. single oral dose
2. repeated oral dose
3. single intraduodenal dose

Dose / conc.:

75 mg/kg bw/day

Remarks:

single oral dose

No. of animals per sex per dose / concentration:

5 (oral administration)
6 (one group was dosed intraduodenally after bile duct fistulation)
Please refer to table 1 in the "Any other information on materials and methods incl. tables" section.

Control animals:

no

Type:

absorption

Results:

The test substance was rapidly absorbed from the gastrointestinal tract into the plasma. Max. equivalent concentration was reached 30 - 70 min after administration.

Type:

distribution

Results:

The test item was rapidly distributed in most organs/tissues after 1 h. Highest concentrations: stomach and small intestine. Lung, heart, kidneys, liver hab in higher concentration than the whole body (fast absorption; early onset of renal excretion).

Type:

excretion

Results:

Only traces of the administered dose (<0.01%) were expired within the test period of 72 h.

Type:

metabolism

Results:

urine: CPT-N-glucuronide, CPT; bile: glucuronic acid conjugate of parent compound hydroxylated in the cyclohexylring; faeces: 4-hydroxy-CPT, cyclohexyl-hydroxylated parent compound (presumably the aglycon of the GA conjugate identified in bile)

Details on absorption:

The test substance seems to be rapidly absorbed from the gastrointestinal tract of the rats in all dose groups after oral administration of a suspension in 0.5% aqueous tragacanth. The plasma concentration values and the plasma curve analysis show that the absorption started immediately after dosing. The maximum equivalent concentration was reached ca. 30 to 70 minutes postadministration in all dose groups.
The dose normalized equivalent concentration peaked at 0.11 and 0.12 for the high dose groupr at 0.66 and 0.56 for the low dose group and at 0.74 and 0.71 for the pretreated rat, i.e. between 11% and 74% of the equidistribution concentration when comparing the average values for five animals in each test. The plasma maximum was independent of sex and pretreatment and not proportional to the administered dose, the latter indicating incomplete absorption and unchanged parent test compound in the faeces. The bile-fistulation test showed that a very high percentage of the test compound at the low dose level was absorbed from the GIT, i.e. at least 98% of the recovered.
Absorption amounted to at least 42% (male animals) and 50% (female animals), given as the renal portion plus the sum of the organs without gastro-intestinal tract, in the high dose experiment. Whereas this amount of absorption in the high dose tests can only be a crude estimate, the absorbed amount is well known for the low dose experiments, where a bile fistulation experiment was performed. In this case the amount absorbed, calculated as the sum of the organs plus bile plus renal radioactivity was 97.9% of the recovered radioactivity (ie 103.5% of the dose).

Details on distribution in tissues:

Plasma: All dose groups showed a fairly quick decrease of the radioactivity concentration after the maximum which was due to very fast distribution and excretion. The menu driven pharmacokinetic fitting programme TOPFIT (Version 2.0) was used to calculate the pharmacokinetic parameters from plasma curve analysis. Linear standard compartment models were used for iterative computations.
The radioactivity concentrations in plasma (arithmetic means from 5 animals) were subjected to three compartment modelling; the correlation coefficients for the fit were higher than 0.99 in all tests. The resulting biokinetic parameters showed a minor to negligible sex dependence. The biokinetic behaviour of the total radioactivity was characterized by medium quick elimination; the corresponding elimination half-lives were rather short and ranged from about 10 to 41 h. In accordance with the ready excretion of the total radioactivity the total clearance was calculated to be very high in all dose groups, i.e. 6.7 to 21.2 [mL/(min*kg bw)] without correction for the bioavailability. The renal clearance (CLR = CL x renal excretion fraction) was rather high with values between 5.3 and 10.4 [mL/(min*kg bw)]. These numbers are in the range of the maximum glomerular filtration capacity of the kidney meaning that the bulk of radioactivity was eliminated via glomerular filtration without reabsorption. The hypothetical distribution volume under steady state conditions (Vss) of the total radioactivity was dependent on the treatment, the low dose animals showing no sex dependence with values of 727% and 907% of the body volume; the high dose animals displayed sex dependence in that for males values of 1040% and for females 477% had been calculated. A still more dramatic difference was to be seen comparing males and females of the pretreated group: males showed a - hypothetical - distribution volume of 585% and females of 244% of the body volume. These values reflect low to medium concentrations of the total radioactivity in the central compartment in comparison with some peripheral compartments. Dose level and pretreatment seem to influence the distribution behaviour of total radioactivity in the body, females are likely to be more susceptible to this influence. The mean residence time MRT of the total radioactive dose in the plasma was short in all dose groups. Values varying between ca. 6 and 14 hours indicate that elimination is a very fast process.

Whole-body autoradiography: The basic results of the plasma curve analysis were confirmed in the whole body autoradiography experiments. In addition to manifesting the rapid absorption, distribution and excretion of the administered radioactivity in the rats, organs and tissues with increased radioactivity concentration were shown. The dose was set at 4.5 mg/kg bw in order to improve the detection limit but still be able to compare the results with the main experiments at a dose of 1.5 mg/kg bw.
The total radioactivity was absorbed rapidly from the gastrointestinal tract (GIT) of the rat. One hour after administration radioactivity appeared in most organs and tissues in different concentrations. The highest concentrations were found in the stomach and the small intestine. The tissues and organs containing high levels of blood like lung, heart volume, kidneys and liver appeared in higher concentration than the whole rat body indicating fast absorption and a very early onset of the renal excretion. Fat, testis and muscle, including the heart muscle, contained about equally low concentrations of radioactivity. Very low radioactivity concentrations were detected in the central nervous system, the eye and the lower part of the large intestine. This phenomenon is probably due to a first pass effect converting the test substance to polar metabolites which do not penetrate the blood-brain barrier. In general, a similar distribution of radioactivity was observed four hours after dosage with a still more pronounced concentration difference between the metabolizing organs and tissues liver and kidney having high radioactivity concentrations and the bone marrow, brain and again the lower parts of the large intestine with nearly no detectable radioactivity. Organs having high concentrations of blood like heart content and lungs appeared above the average of the rats body, thus indicating high blood circulation of radioactivity and giving a hint to some enterohepatic circulation In combination with the still high concentration in liver.
Eight hours postadministration the intensity of all organs and tissues except stomach and intestine and to a minor extent liver and kidney was very low. Low concentrations of radioactivity were still detectable in blood (heart contents) and lung. The intensity in the central nervous system had decreased below the detection limit due to the fact that a large amount of the radioactivity had moved into the large intestine or been excreted.
At 24 hours after dosing no residues were detected in the rat body with the exception of intestine, liver and kidney. The concentration in the intestine was highest, the radioactivity in liver was much weaker and the intensity of the renal cortex was just barely increased above background. The same picture was obtained 48 after administration. The high concentration in liver in combination with the GIT at late stages of the investigation demonstrates the existence of an enterohepatic circulation of the compound derived radioactivity. The rapid excretion of the test substance and its metabolites led to a quick depletion of all organs and tissues except for liver and kidney within 48 hours after administration.

Details on excretion:

In a preliminary test the expiration of CO2 and other labelled volatiles was followed over a period of 72 hours using male rats. Only traces of the administered dose (<0.01%) were expired within the test period of 72 h. This result clearly showed that the selected labelling position in the molecule was stable concerning metabolism in vivo.
After oral administration on average more than or equal to 99.8% of the recovered radioactivity was excreted via urine and faeces within 48 hours for all dose groups. The major route of excretion was renal for the low dose groups (ratio renal to faecal: approx. 7 - 8 ) irrespective of the gender; the high dose group showed an about equal ratio of renal and faecal elimination (0.7 to 1.0); the pretreated group again had a preferentially renal excretion (ratio of about 5).
The rate of renal and faecal excretion was very high. Within 48 h following administration the radioactivity was excreted via these routes to a great extent (> 94% of the dose in all tests). Bile-cannulated animals excreted the radioactive dose very fast, too. More than 85% (99%) of the biliary excretion occurred in the first 1 h (12 h) postadministration. This is a result of the fast absorption and metabolism as described above. In the bile-fistulation test only 2.2% of the dose was excreted with faeces.
The percentage of renal excretion was different between intact and bile-fistulated animals: 88% and 59% of the recovered radioactivity, respectively. It is presumed that this difference was due to enterohepatic circulation of compound related radioactivity. Portions of the biliarily excreted radioactive dose were reabsorbed from the gut and redistributed to liver and kidney prior to final elimination.

The radioactivity remaining in the body without the gastrointestinal tract was in all cases less or equal to 0.14% and in the low dose tests below 0.1% of the dose at sacrifice after oral administration.
From the kinetics of the renal and faecal excretion as well as from the elimination kinetics of the total radioactivity from plasma it can be concluded that these small amounts of radioactivity were subject to further elimination from the body. Among the organs examined for radioactive residues only liver and to a minor extent kidney retained a significant amount of the dose. This residue was very low and reached 0.14% of the dose at most. Among 15 organs and tissues checked only liver in the high dose group was found to contain a concentration of total radioactivity higher than P = 0.01, i.e. 1% of the equidistribution concentration. It ranged from 0.005 to 0.015 in all dose groups. The elevated concentration in the liver support the assumption that this organ was the main metabolizing site in the rat for the test substance.

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 35.2 h

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 40.6 h

Test no.:

#5

Toxicokinetic parameters:

half-life 1st: 14.8 h

Test no.:

#6

Toxicokinetic parameters:

half-life 1st: 9.67 h

Test no.:

#7

Toxicokinetic parameters:

half-life 1st: 37 h

Test no.:

#8

Toxicokinetic parameters:

half-life 1st: 10 h

Test no.:

#2

Toxicokinetic parameters:

AUC: 2.75

Remarks:

µg/(gxh)

Test no.:

#4

Toxicokinetic parameters:

AUC: 2.26

Remarks:

µg/(gxh)

Test no.:

#5

Toxicokinetic parameters:

AUC: 58.8

Remarks:

µg/(gxh)

Test no.:

#6

Toxicokinetic parameters:

AUC: 95.5

Remarks:

µg/(gxh)

Test no.:

#7

Toxicokinetic parameters:

AUC: 2.58

Remarks:

µg/(gxh)

Test no.:

#8

Toxicokinetic parameters:

AUC: 3.73

Remarks:

µg/(gxh)

Test no.:

#2

Toxicokinetic parameters:

Cmax: 0.856

Remarks:

µg/g

Test no.:

#4

Toxicokinetic parameters:

Cmax: 0.661

Remarks:

µg/g

Test no.:

#5

Toxicokinetic parameters:

Cmax: 8.16

Remarks:

µg/g

Test no.:

#6

Toxicokinetic parameters:

Cmax: 8.94

Remarks:

µg/g

Test no.:

#7

Toxicokinetic parameters:

Cmax: 1.07

Remarks:

µg/g

Test no.:

#8

Toxicokinetic parameters:

Cmax: 1.04

Remarks:

µg/g

Metabolites identified:

yes

Details on metabolites:

According to chromatographic analyses of the rat excreta the test substance was metabolized extensively. Unchanged parent compound was detected only as a minor component in faeces extracts of the low dose groups and as main component in faeces extracts of the high dose male rats and to a lesser extent in those of the female animals. No parent compound was detected in composite urine of the tests nor in bile. The identity of the parent compound excreted with the faeces was confirmed by HPLC. In urine the two major components were isolated. Successive preparative HPLC of the relevant metabolites finally gave fractions suitable for spectroscopic investigations. For confirmation with a greater amount of material these preparations were repeated. The structures emerging from spectroscopic structure elucidation were CPT-N-glucuronide (code ECW10217) for fraction 3 and CPT for fraction 4. From bile the major component was isolated and purified by successive preparative HPLC to give a metabolite fraction suitable for structure elucidation. The structure found for the compound with the code ECW10212 by spectroscopic interpretation was that of a glucuronic acid conjugate of parent compound hydroxylated in the cyclohexylring.
Two more trace metabolites found in faeces extracts were identified by co-chromatography and matching of retention times in the above mentioned two HPLC systems.
Quantification: The metabolites were determined quantitatively in the pooled urine and in the bile samples and in extracts from faeces containing more than or equal to 98% of the total radioactivity in the respective matrix for each dose group and sex. For quantitation the reverse phase HPLC-method YRC2A with gradient elution was used.

Urine: The chromatographic profiles of all composite urine samples were dominated by two prominent peaks, which had been identified as CPT and CPT-glucuronide. These two identified metabolites together made more than 91% of the urine fraction analysed in the low dose groups, more than 74% in the high dose groups, and more than 82% in the pretreated tests. The glucuronide showed the greater amount in the chromatograms, ranging from 47% to 76%, except for the low dose female animals, where the free CPT had 49% and the glucuronide only 42% of the radioactivity analysed. The sum of the unidentified portion ranged from 8% of the analysed in the low dose males to 26% in the high dose females, but no single unidentified compound contained more than 4% of the dose.

Faeces: In the low dose group only 11% and 12% of the recovered radioactivity was; found in tha faeces of the collection period 0 - 24 h, and negligible amounts in the period 24 - 48 h. 44% or 55% of the main fraction (ie 4.5% and 6.1% of the recovered radioactivity) was extracted and sufficiently freed from matrix material to be analysed. In the high dose group 57% and 43% of the recovered radioactivity was found in the 24 h-faeces, and 1% to 6% in the period 24 - 48 h. 82% to 67% of the main fraction (ie 47% and 33% of the recovered radioactivity) was extracted and suitable for analysis. Parent compound was the major component in all faeces extracts analysed; it ranged from 14% to 62% of the radioactivity analysed, the amount relative to the totally recovered radioactivity with 30% and 9% being considerably higher in the high dose tests 5 and 6 than in the low dose tests 2 and 4 (1.6% and 0.6%). The second important metabolite in faeces extracts was CRT either free or as glucuronide. Minor amounts (below 2%) in the range of traces were found as 4-hydroxy-CPT and - except for high dose-male animals - cyclohexyl-hydroxylated parent compound. Single unidentified compounds contained not more than 1% of the dose in the low dose tests and not more than 3% in the high dose tests.

Bile: Bile collected in the bile fistulation experiment from 0 to 1 h contained more than 88% of the fraction of the dose eliminated with the bile and analysed directly. Several metabolites were separated with the HPLC system YRC2A that was used in this analysis. Among these three compounds were identified and quantified: CPT was the main metabolite with 12% of the radioactivity analysed (4.3% of the dose), followed by the glucuronide of the cyclohexyl-hydroxylated parent compound with 11% (3.7% of the dose); CPT-glucuronide was only a minor metabolite with 4% (1.5% of the dose).

Total rate of identification: The total rate of identification was rather high in the low dose group; it covered 79% to 87% of the administered dose (83% to 82% of the total recovered). The identification was lower in the pretreated group (69% to 65% of the dose; 69% of the recovered), and lowest in the high dose group (65% to 51% of the dose and 67% to 52% of the recovered). The lower identification in the pretreated group is due to the fact that the faeces extracts were not analysed. Biotransformation following high dose treatment gives on the one hand much higher amounts of unabsorbed parent compound, but leads on the other hand to higher rates of metabolisation with many minor metabolites which were not identified. In all tests the sum of CPT and its glucuronic acid conjugate were the main metabolites, females always showing more of the free CPT than the appropriate males. In the high dose tests parent was a major compound, too. The two hydroxylated metabolites found exclusively in faeces were minor to trace metabolites with higher relative amounts in the high dose tests.

Influence of the different test conditions:

Dose: There was one major effect attributable to dose variations: whereas after single or multiple oral administration of the low dose the ratio renal to faecal excretion was about 7:1, or 5:1, respectively, it changed to 1:1 in the high dose groups. Also the level of radioactive residues was affected in that in the high dose groups the residues were about twice the ones in the low dose groups, although they were not higher than 0.2% in total. In comparing the low dose and high dose tests one phenomenon is observable: whereas the male animals of the high dose test 5 showed in the majority of the organs and tissues a higher relative (dose-normalized) concentration as compared to males (or females) in test 2 (or test 4) the high dose females showed a much lower relative concentration than the low dose ones. Generally, high dose males obviously exhibited a higher absorption than would be predicted for dose-proportionality whereas high dose females showed a lower one.

Bile-cannulation: Bile-cannulation dramatically influenced the excretion pattern of total radioactivity. The ratio renal to faecal excretion changed to about 27:1 with much lower absolute values than in 'intact' animals. The biliary elimination of close to 40% of the recovered radioactivity together with the renal excretion of 59% was the proof of nearly total absorption of the radioactive dose in male rats.

Sex/Single versus multiple dosing: No significant effect could be observed.

Description of key information

The target substance was rapidly absorbed after oral ingestion with absorption rates of at least 42 - 98% in a dose-dependent manner. More than 90% of the recovered radioactivity was excreted within 48 to 72 h via faeces and urine, with renal excretion being the major elimination route. Extensive metabolism mostly in the liver was determined. Further, the test substance seems to undergo enterohepatic circulation.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Several in vivo studies have been performed to evaluate the toxicokinetic behaviour of the target substance. In detail, the following experimental data are available:

 

A GLP-conform biokinetic behaviour and metabolism study in Wistar rats was performed according to EPA Pesticide Assessment Guidelines (1997a, M-003976-01-1).

[Cyclohexyl-14C] labelled target substance was administered orally (single administration) to groups of 5 rats at a dose of 1.5 (males and females) and 75 mg/kg bw/day (males).

Absorption, distribution and excretion of the test substance in expired air, urine including cage washes, faeces, blood, plasma, serum, organs and tissue was determined 48 h and 72 h after administration. In addition, metabolites in urine (24 h and 48 h after oral administration) and faeces (24 h after administration) were determined by HPLC-MS-MS. Moreover the expired CO2 was trapped and radio-assayed.

Absorption from the gastrointestinal tract started immediately after administration and reached the peak plasma level after 90 to 180 minutes in all cases. The dose normalized equivalent concentrations in plasma were rather high in the low dose groups amounting to 54% to 78% of the equidistributional concentration but low in the high dose groups not exceeding 14% of the equidistributional concentration. In the high dose group absorption was at least 44% in males and >80% in low dose male and female rats.

Recoveries of radioactivity were found to be between 93% and 107% of the dose on average in all experiments.

Biotransformation to volatile metabolites including CO2 was negligible as it was less than 0.04% of the administered dose. 92.7% or more of the recovered radioactivity was excreted within 48 or 72 h after oral administration. The major route of elimination was renal for the low dose groups (76% to 78% of the recovered for the single low dose groups) whereas the high dose group animals showed a higher faecal elimination of radioactivity (55%) as compared to the renal (41%).

No significant difference in the excretion pattern between male and female rats could be observed, but females showed significantly higher residues in the carcass at sacrifice. Comparing single low dose with single high dose, the dose level caused significant effects in the excretion as well as in the residue pattern.

The radioactivity remaining in the body (excluding the gastrointestinal tract) at sacrifice 72 h or 48 h after administration was in the range of 4.4% to 4.6% of the recovered for tests 1 and 2, both low dose males, 7.3% for low dose females after 48 h and 3.0% for high dose males after 48 h. Liver followed by kidneys - and lungs in the low dose groups – containing significant amounts of radioactivity, while gonads, central nervous system and muscle contained the lowest concentrations of radioactivity. According to chromatographic analyses of the excreta the test substance was metabolized by the rats to a very high extent in the low dose experiments; in the high dose tests only the absorbed amount was extensively metabolised resulting in identical biotransformation products. No unchanged parent compound was found in urine neither in low nor in high dose tests. In extracts of faeces parent compound was one of the major radioactive compounds only in the high dose tests. In the low dose tests parent compound was only a minor component in faeces.

One major group of metabolites was identified in all dose groups: cyclohexyl-ethylamine, hydroxylated in different positions of the ring (HO-CEA) leading to several separated, isolated and identified metabolites. Another two prominent metabolites were cyclohexylethylamine (CEA) accounting for 11% to 18% and cyclohexylamine (CA) for 12% to 13% in the low dose animals. Females showed a slightly different pattern with less hydroxylated and more unchanged CEA and CA. The high dose males exhibited a proportionally lower amount of metabolites but approximately 19% of unchanged parent compound. 6 minor important but nonetheless identified metabolites were found, belonging to two groups, one baring the CEA-carbamoyl moiety linked to amino groups of endogenous acids (hydroxyglutamic and tauric acid) and the other one baring the CA-carbamoyl group linked to the nitrogen of ammonia, urea and the sulfur of mercapturic acid. The structure of CEA-CO-glutamic acid is tentative since the spectra were not decisively evaluable due to matrix impurities. The main pathway of biotransformation proceeded via hydrolytical cleavage between the chlorophenyltetrazolinone (CRT) and the N-ethyl-cyclohexyl carbamic acid parts of the molecule.

 

 

A second GLP-compliant toxicokinetic study is available in which absorption, distribution, metabolism, excretion and biotransformation was evaluated in Wistar rats after treatment with phenyl-UL-14C- labelled target substance according to the EPA Pesticide Assessment Guidelines, Subdivision F, 40 CFR Part 158, 85-1 (1982) (1997b, M-003977-01-1). The test compound was administered orally to 3 groups of five male and female rats each. One group of six animals was dosed intraduodenally after bile duct fistulation. The objective of this study was to study the dependence of dose (1.5 and 75 mg/kg bw; single oral dose), pretreatment (1.5 mg/kg bw; repeated oral dose: non-radioactive compound once per day and 24 h after the 14th non-radioactive dose a single radioactive dose) and sex. The intraduodenal single dose was 1.5 mg/kg bw. In a special test the expired CO2 was trapped and radio-assayed in addition to collecting the excreta for radioassays. For this purpose male rats received a single oral dose of 1.5 mg/kg bw. The dose was prepared by suspending the target substance (or the radiolabeled compound) in 0.5% aqueous tragacanth.

Time dependent distribution of the radioactivity in the whole rat body was followed in an ancillary test. For this purpose a group of male rats was given a single oral dose of 4.5 mg/kg bw. Whole body autoradiographs (WBA) of rat sections cut at distinctive planes were prepared and evaluated qualitatively. In finding the dose, the specific radioactivity, the toxicity of the test compound and the stability in the administration solution were taken into account. The study followed the dosage recommendations of the Pesticide Assessment Guidelines of the U.S. Environmental Protection Agency (EPA). The total radioactivity in plasma and excreta was quantified in dependence on time. The radioactivity in organs and tissues was determined at sacrifice 48 and 72h post administration. These data served as basis for understanding the pharmacokinetic behaviour of the parent compound and/or its labelled metabolites in the rat. Thus a mathematical description of the absorption, distribution and excretion, i.e. the "biokinetic profile" of the total radioactivity was possible.

The terminal biotransformation products in the rat were characterized, identified, and quantified in urine, faeces and bile dependent on dose, sex, and non-radioactive pre-treatment.

Absorption from the gastrointestinal tract started immediately after administration and reached the peak plasma level after 40 to 60 minutes in all cases. The dose normalized equivalent concentrations in plasma were rather high in the low dose groups amounting to 56% to 74% of the equidistribution concentration but low in the high dose groups not exceeding 12% of the equidistribution concentration. The absorption of the test substance amounted to at least 42% (male animals) and 50% (female animals), given as the renal portion plus the sum of the organs without gastro-intestinal tract, in the high dose experiment. In the low dose group, the amount absorbed, calculated as the sum of the organs plus bile plus renal radioactivity was 97.9% of the recovered radioactivity (i.e. 103.5% of the dose).

The rapid absorption of radioactivity was confirmed by whole-body autoradiography. Recoveries of radioactivity were found to be higher than 94% of the dose on average in all experiments. For better comparison of tests the percentages presented in the report were also normalized for recovered radioactivity. Biotransformation to volatile metabolites including CO2 was negligible, i.e. less than 0.005% of the administered dose. 99.8% or more of the recovered radioactivity was excreted within 48 or 72 h after oral administration. The major route of elimination was renal for the low dose groups (87% to 89% of the recovered for the single low dose groups; 83% to 84% for the pretreated low dose groups) whereas the high dose group animals showed a nearly equal distribution of radioactivity to faeces and urine. Statistical analysis of the results revealed the following effects:

- There was no significant difference in the excretion pattern between male and female rats;

- Dose variation caused significant effects in the excretion as well as in the residue pattern, comparing single low dose with single high dose and multiple low doses to single high dose;

-There was no significant difference in the excretion and retention behaviour between single and multiple low dosing.

The radioactivity remaining in the body excluding the gastrointestinal tract at sacrifice 48 h after administration was in the range of or below 0.2% of the recovered. Liver followed by kidneys were the only organs or tissues containing significant amounts of radioactivity; gonads, central nervous system and muscle contained the lowest concentrations of radioactivity. According to chromatographic analysis of the excreta, the test substance was metabolized extensively by the rats. No unchanged parent compound was found in urine neither in low nor in high dose tests. In extracts of faeces parent compound was one of the major radioactive compounds only in the high dose tests. In the low dose tests parent was only a minor component in faeces. In the ancillary bile cannulation experiment no parent was detected in native bile. Two major metabolites were identified in all dose groups: the hydrolysis product of parent compound containing the radio-labelled moiety, CRT, either free or conjugated with glucuronic acid. Spectroscopic investigations revealed that the binding position was the nitrogen in 4-position of the tetrazolinone-ring. Two minor metabolites were identified and quantified, i.e. the 4-hydroxy-CPT and cyclohexyl-hydroxylated parent compound, both found exclusively in faeces extracts. The latter was also found as glucuronic acid conjugate in the bile of the bileduct-fistulated animals. The main pathway of biotransformation proceeded via hydrolytical cleavage between the chlorophenyl-tetrazolinone (CRT) and the N-ethyl-cyclohexyl carbamic acid parts of the molecule. The unlabelled cyclohexyl part was no longer followed, and the CPT-part was finally excreted either free or conjugated.

 

A supporting GLP-conform study was performed investigating the distribution of the target substance and its metabolites in selected organs and tissues of Wistar rats under the recommendations of the MAFF (Japan) (1997c, M-003972-01-1). Cyclohexyl-1-14C- and the chlorophenyl-UL-14C-labelled compounds were administered orally to 3 groups of five male rats, each. The dose was prepared by suspending the target substance in 0.5% aqueous tragacanth. The dose was 1.5 mg/kg body weight (bw) as was used in the basic biokinetic and metabolism studies described above.

Total radioactivity in plasma and excreta was quantified at sacrifice. The sacrifice times were 60, 120 and 360 minutes for the tests with [cyclohexyl-1-14C] labelled target substance reflecting the increasing phase, peak and decreasing phase of the plasma level as was determined in a previous basic study; for the tests with [chlorophenyl-UL-14C] labelled target substance the corresponding sacrifice times were 30, 60 and 180 minutes. Radioactive metabolites identified and characterized in previous basic studies were used as reference compounds for identification of biotransformation products in the presented study. Metabolites not available were isolated, identified and then used as references as well.

In the tests with the cyclohexyl-labelled test item one major group of metabolites was identified in all groups: cyclohexyl-ethylamine, hydroxylated in different positions of the ring (HO-CEA) leading to several separated, isolated and identified metabolites (the position of the hydroxyl group in the cyclohexyl-ring was not elucidated). Its amount in plasma was relatively stable around 4% of the total radioactive residues (TRR) in plasma, whereas in liver and kidney it dropped from 18% and 23% to 5% and 9% at sacrifice times of 60 min and 360 min. Another two prominent metabolites were cyclohexyl-ethylamine (CEA), accounting for 1% to 3% in plasma, 4% to 5% in liver and 15% to 28% in kidney, and cyclohexylamine (CA) which accounted for 21% to 3% in plasma, 6% to 2% in liver and 14% to 4% in kidney. Three minor important but nonetheless identified metabolites were found, belonging to two groups, one baring the CEA-carbamoyl moiety linked to the amino group of glutamic acid and the other one baring the CA-carbamoyl group linked to the nitrogen of urea and the sulfur of mercapturic acid (CA-CO-S-cys-N-ac). The latter was in the initial test the most prominent metabolite in urine (30%), then dropping to 14% of the renal radioactivity. It showed, in parallel to the fate in urine, also rapid decrease in all organs, in plasma dropping from 1.1% to 0.3% of the TRR, in liver from 1.7% to 0.2% and in kidney from 5.9% to 0.5%. This behaviour of the metabolite gave rise to confirmation of the structure of this metabolite by spectroscopic investigations after chromatographic isolation.

In the tests with chlorophenyl-labelled test item only two main metabolites were found, chlorophenyltetrazolinone (CPT) and its glucuronic acid conjugate. In plasma both together accounted for 94% to 79% from the sacrifice times 30 min to 180 min after oral administration. The conjugate showed a slight increase from 47% to 52% and the free CPT dropped from 47% to 28%.

In liver both were nearly constant with amounts of 72% to 67% for the conjugate and 5% to 4% for CPT; a minor metabolite was detected in liver which was already known from bile investigations in a previous basic study: the glucuronic acid conjugate of hydroxylated parent compound in amounts of 4% to 5%.

In kidney behaviour identical to plasma was found in that the conjugate increased slightly from 43% to 56% and free CPT dropped from 54% to 36%; the sum of both remained nearly constant, i.e. from 96% to 92%. The recoveries varied from 77% to 104%, mostly above 80% of the dose. The balances were somewhat affected by the fact that the total radio-activities of the organs liver and kidney were only determined as the sum of the radioactivity in the extracts and the radioactive unextracted solids. The main pathway of biotransformation proceeded via hydrolytical cleavage between the chlorophenyltetrazolinone (CPT) and the N-ethyl-cyclohexyl carbamic acid parts of the molecule.

 

In addition, a supporting GLP-conform kinetic behaviour study was performed in female rats (Hsd Cpb:WU) with two radiolabelled test items, cyclohexyl-1-14C- and chlorophenyl-UL-14C-labelled target substance (1998a, M-003970-01-1). Each test compound was tested orally in 4 groups of three animals at a dose of 1.5 mg/kg bw. The four groups differed in pre-treatment either with 50 ppm or 6400 ppm in the feed for 3 days or for 13 weeks with unlabelled test substance. Plasma was collected in dependence on time after the radioactive administration, in short intervals at the beginning and longer ones later-on, and radio-assayed. Urine was collected in the intervals 0 - 4h, 4 - 8h, 8 - 24h, 24 - 48 and 48 - 72h and faeces daily, and radio-assayed. Urine was analysed directly by HPLC and faeces after extraction. In the tests with the [cyclohexyl-1-14C] target substance the plasma concentrations were very different from those found in the tests with [chlorophenyl-UL- C] target substance. The differences between the tests are on one hand typical for an induction of metabolic processes as a consequence of pre-treatment, and on the other hand exhibit that, despite of some induction to be supposed, an endogenous supply of compounds necessary for complete metabolism seems to be exhausted in the tests with the high-dose-pre-treated animals. This effect leads to relatively low plasma concentrations and even lower ones in the tests with the animals pre-treated for 13 weeks. Radio-assays and chromatographic analyses of the metabolites did not show new metabolic or degradation ways as already reported in previous studies. The differences of the radiolabelled test items were quantitative in nature, showing with both labels induction of metabolic processes which were easily recognizable from either the plasma concentrations (in the tests with [cyclohexyl-1-14C]-labelled target substance) or the metabolic pattern ([chlorophenyl-UL-14C]-labelled target substance) in the tests with the animals pre-treated with the low dose for 13 weeks. The metabolic and biokinetic behaviour of the target substance labelled in two sites of the molecule is reported above. In these studies complete absorption and total cleavage of the molecule in two parts with subsequent separate biotransformation has been demonstrated. In all tests with the chlorophenyl-labelled compound the main metabolites were CRT and its glucuronide, as was also found in this study. Since there is no additional phase I metabolism necessary for elimination, only the supply of UDP-glucuronic acid for the conjugation step could be enhanced by the pre-treatment, which was obviously found in the pre-treated animals as shown by the faster and somewhat higher plasma peak.

 

There are no studies available in which inhalation or dermal absorption were investigated. In accordance with Annex VIII, Column 1, Item 8.8 of Regulation (EC) 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2017), dermal and inhalative absorption are assessed to the extent that can be derived from the relevant available information on physicochemical and toxicological characteristics.

 

The test substance is a colourless crystalline solid with a molecular weight of 349.9 g/mol. The substance has a low vapour pressure of 5.0E-10 hPa at 20 °C and 1.0E-9 hPa at 25 °C. The log Pow was determined to be 3.6 at 20°C and the water solubility is 2.5 mg/L at 20°C. The median particle size distribution measured with laser diffraction was L50= 49.0 µm, particle size L10= 20.9 µm and particle size L90= 293.9 µm. The surface tension was determined to be 66 mN/m at 20 °C, 1.8 mg/L of test substance in water and 69 mN/m at 20 °C, 0.7 mg/L of test substance in water. Furthermore, the test item has no acidic or basic properties in aqueous solutions. It is not possible to specify dissociation constants for water.

 

Absorption

Absorption is a function of the potential of a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, the octanol/water partition coefficient (log Pow) value and the water solubility. The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2017).

 

Dermal absorption

There are no experimental data available on dermal absorption of the test substance. On the basis of the following considerations, the dermal absorption of the substance is considered possible but to rather limited extent. The test substance is a solid, which does not favour dermal absorption, since dry particulates will have to dissolve into the surface moisture of the skin before uptake can begin. However, the molecular weight of the substance indicates possible dermal uptake (100 g/mol < test substance (349.9 g/mol) < 500 g/mol). Furthermore, the log P of 3.6 might favor dermal absorption, although the water solubility of 2.5 mg/L indicates a rather low absorption potential through the skin.

Since the surface tension of the test substance in an aqueous solution is > 10 mN/m, the substance is not a surfactant and thus dermal uptake is not enhanced.

The dermal permeability constant Kp of the substance was estimated to be 0.00421 cm/h using DermwinTM and taking into account the log Pow and molecular weight. Furthermore, the maximum flux Imax (Imax = Kp [cm/h] x water solubility [mg/cm³]) was calculated to be 0,010525 µg/cm²/h. This flux value can be assigned to a possible but low dermal absorption.

The repeated-dose dermal toxicity study performed in rats (1997k) contributes the assumption that the test substance is absorbed following dermal contact as a treatment-related decrease in Cholinesterase activity was observed in erythrocytes at the highest dose level (1000 mg/kg bw/day) tested. In an acute dermal toxicity study in rats, a LD 50 > 5000 mg/kg bw was determined (1995 b). In the absence of adverse effects, no conclusion can be drawn from this study regarding toxicokinetic behaviour, as absence of effects could be due to either low hazardous properties of the test substance or limited dermal absorption. As the test substance was not irritant to skin (1995c), dermal absorption will not be enhanced due to damage to skin surface. In summary, based on the physicochemical and toxicity data, it can be anticipated that dermal absorption of the test substance occurs, but most probably to a rather low extent.

 

Inhalative absorption

There are no data available on inhalative absorption of the test substance. Substances that can be inhaled include gases, vapours, liquid aerosols (both liquid substances and solid substances in solution) and finely divided powders/dusts (ECHA, 2017). In general, depending on the physicochemical properties, test substances may deposit in the respiratory tract followed by absorption and/or transport out of the respiratory tract with possible subsequent uptake via the oral route. Since the test substance has a very low vapour pressure of 5.0E-10 hPa at 20 °C and 1.0E-9 hPa at 25 °C, it has a low volatility. Therefore, under normal use and handling conditions, inhalation exposure and thus availability for respiratory absorption of the substance in the form of vapours, gases, or mists is not significant. However, in humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract (ECHA, 2017). Based on the median particle size distribution (L50= 49.0 µm, L10= 20.9 µm and L90= 293.9 µm), test substance particles may reach the thoracic region once they have been inhaled. The moderate Log p of 3.6 and the relatively low water solubility of 2.5 mg/kg bw favors absorption across the respiratory tract epithelium by passive diffusion. Furthermore, highly lipophilic compounds with a Log P >4 may be taken up by micellular solubilisation. It is possible that poorly water-soluble dusts, deposit in the nasopharyngeal region and could be coughed or sneezed out of the body or swallowed. Dusts depositing in the tracheo-bronchial region would mainly be cleared from the lungs by the mucocilliary mechanism and swallowed, but a small amount may be taken up by phagocytosis.

In an acute inhalative toxicity study in rats, a LC 50 > 5085 mg/m³ air was determined (1996b). In this study, no mortality or clinical signs of toxicity were observed. However, a decrease of body weight was noted and there was a concentration-dependent effect on body temperature observed in all dose groups exposed to the test compound, indicating that absorption of the test substance following inhalation has occurred. In conclusion, the physicochemical and toxicity data indicate that inhalative absorption of the test substance seems to be possible.

 

Overall, the toxicokinetic studies performed with the target substance indicate a rapid absorption in the gastrointestinal tract of the target substance after oral administration followed by metabolism and distribution within the body. Based on physicochemical and toxicity data, it can be anticipated that the test substance is also absorbed via the dermal and inhalative route. However, dermal absorption is anticipated to be rather low whereas and inhalative absorption is not considered to be higher than through the intestinal epithelium.

After oral ingestion, the liver followed by kidneys and lungs contained significant amounts of radioactivity, while gonads, central nervous system and muscles contained the lowest concentrations of radioactivity. After administration of [Cyclohexyl-14C] labelled target substance, one major group of metabolites was identified in all dose groups: cyclohexyl-ethylamine, hydroxylated in different positions of the ring (HO-CEA) leading to several separated, isolated and identified metabolites. Another two prominent metabolites were cyclohexylethylamine (CEA) accounting for 11% to 18% and cyclohexylamine (CA) for 12% to 13% in the low dose animals. 6 minor important but nonetheless identified metabolites were found, belonging to two groups, one baring the CEA-carbamoyl moiety linked to amino groups of endogenous acids (hydroxyglutamic and tauric acid) and the other one baring the CA-carbamoyl group linked to the nitrogen of ammonia, urea and the sulfur of mercapturic acid. Administration of phenyl-UL-14C- labelled target substance resulted in the identification of two major metabolites: the hydrolysis product of parent compound containing the radio-labelled moiety, CRT, either free or conjugated with glucuronic acid. Two minor metabolites were identified and quantified, i.e. the 4-hydroxy-CPT and cyclohexyl-hydroxylated parent compound, both found exclusively in faeces extracts. Further, exposure to cyclohexyl-1-14C labelled target substance resulted in one major group of metabolites: cyclohexyl-ethylamine, hydroxylated in different positions of the ring (HO-CEA) leading to several separated, isolated and identified metabolites (the position of the hydroxyl group in the cyclohexyl-ring was not elucidated). Another two prominent metabolites were cyclohexyl-ethylamine (CEA) and cyclohexylamine (CA). Administration of chlorophenyl-UL-14C-labelled target substance led to two main metabolites namely, chlorophenyltetrazolinone (CPT) and its glucuronic acid conjugate. Finally, renal elimination was identified as major elimination route.