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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:

Not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Objective of study:

excretion

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

A single oral dose of 1500 mg/kg bw 14C labelled test material was administered to six male rats. Urine samples were taken at the conclusion of 0-24, 24-48 and 48-72 sampling intervals. The quantity of parent compound-equivalents was measured by LSC and expressed as percentage of administered dose. Metabolites were isolated and purified through the use of TLC and HPLC and identified by high resolution NMR spectroscopy and MS.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilington, MA, USA
- Weight at study initiation: 142-163 g
- Fasting period before study: 18 hours
- Housing: Nalgene metabolism cages
- Individual metabolism cages: yes
- Diet: Charles River Rat, Mouse and Hamster formula ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1400 mg unlabelled test material was mixed with 12.6 mg labelled test material in 1 mL hexane. After mixing, the hexane was evaporated and the residue dissolved in 3 mL corn oil and mixed. Each animals was administered approximately 0.7 mL of dosing solution.

Duration and frequency of treatment / exposure:

Once

Dose / conc.:

1.5 other: g/kg

Control animals:

yes, concurrent vehicle

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: 24, 48 and 72 hours after dosing.

Details on excretion:

Of the applied dose, 12 % was excreted in the urine.

Metabolites identified:

yes

Details on metabolites:

HPLC radiochromatograms generated for the 0-24, 24-48 and 48-72 hour urine samples all qualitatively resemble each other in that there are five major zones of radioactivity. Moreover, the HPLC profiles are qualitatively the same as profiles obtained in an earlier oral dose study and 13 week dietary study (Banijamali and Tortora, 1998a) in spite of different dose levels and exposure regimes. In addition, the parent compound was not detected in the urine.

The molecular weights of metabolites 1 to 5 were determined to be 294, 382, 296, 296 and 280. In order of increasing polarity the following urinary metabolites were identified:

Metabolite 5: 1-[4-{1.1-dimethyl-2-hydroxyethyl)phenoxy]-2,x-cyclohexane-diol (HOMe-TBOC-diol)
Metabolite 4: 1-[4-{1.1-dimethyl-2-hydroxyethyl)phenoxy]-2,x,x'-cyclohexane-triol (HOMe-TBOC-triol)
Metabolite 3: 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,4,5-cyclohexane-triol (HOMe-TBPC-triol)
Metabolite 2: 1-[4-(2,x-dihydroxycyclohexoxy)phenyl]-2,2-dimethyl sodium sulphate (HOMe-TBPC-diol sulphate)
Metabolite 1: 1-{4-(2,x-dihydroxycyclohexoxy)phenyl]-2,2-dimethyl acetic acid (Carboxy-TBPC diol)

Conclusions:

Interpretation of results: bioaccumulation potential cannot be judged based on study results
Urinary HPLC radiochromatograms taken over time and mass spectral analysis indicate that the test material is rapidly and completely metabolised in male rats to five major metabolites of greater polarity than the parent compound. The data indicate a metabolic pathway that involves hydrolysis and hydroxylation of the parent compound to 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,x-cyclohexane-diol (HOMe-TBPC-diol). This metabolite is then subject to either sulphate conjugation to give 1-[4-(2,x-dihydroxycyclohexoxy)phenyl-2,2-dimethylethyl sodium sulphate (HOMe-TBPC-diol sulphate) or further oxidation to give 1-[4-(2,x-dihydroxycyclohexoxy)phenyl]-2,2-dimethyl acetic acid (Carboxy-TBPC-diol) or hydroxylation to give 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,4,5-cyclohexane-triol (HOMe-TBPC-triol) and 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,x,x'-cyclohexane-triol (HOMe-TBPC-triol). Moreover, the same degradation pathway is apparent regardless of dose level or route of administration.

Executive summary:

Male rats were treated with an oral dose (1.5 g/kg) of ¹⁴C-test material. Urine and faeces were collected for 72 hours after treatment. During this time, 12 % of the applied dose was excreted in the urine. Five major metabolites were detected by HPLC and identified by high resolution FT-NMR and MS. Spectral analysis indicated that test material was rapidly degraded by rats to more polar products and metabolism of cyclohexyl ring is strongly favoured.

In order of increasing polarity, the following metabolites were isolated in rat urine and identified as:

1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2, x-cyclohexane-diol

1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,x,x'-cyclohexane-triol

1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,4,5-cyclohexane-triol

1-[4-(2,x-dihydroxycyclohexoxy)phenyl]-2,2-dimethylethyl sodium sulfate

1-[4-(2,x-dihydroxycyclohexy)phenyl]-2,2-dimethyl acetic acid

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Isolation of the sixth urinary metabolite found in the female rat through the use of HPLC and NMR.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

From Kehoe (1988):

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Housing: individually in screen bottom stainless steel cages (metabolism cages for the kinetic phase)
- Diet: Purina Rodent Laboratory Chow® #5002 ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 ºF
- Humidity: 50 ± 20 %
- Photoperiod: 12 hours light/12 hours dark

IN-LIFE DATES: From: 5th May 1987 To: 28th August 1987

Duration and frequency of treatment / exposure:

Once

Metabolites identified:

yes

Details on metabolites:

An HPLC radiochromatogram revealed the characteristic five major zones of radioactivity plus the sixth female-unique metabolite. This sixth metabolite was the most polar of all metabolites and was identified as 1-[4-(2,4,5-trihydroxycyclohexoxy)phenyl]-2,2-dimethyl acetic acid (Carboxy-TBPC-triol).

Conclusions:

Interpretation of results: bioaccumulation potential cannot be judged based on study results
A urinary HPLC radiochromatogram confirmed the presence of a sixth urinary metabolite that is unique to female rats. This metabolite, the most polar of all six female metabolites, has been identified as 1-[4-(2,4,5-trihydroxycyclohexoxy)phenyl]-2.2-dimethyl acetic acid (Carboxy-TBPC-triol).

Executive summary:

The sixth urinary metabolite of test material found in the female rat was isolated and identified through the use of HPLC and NMR. Spectral analysis gave an indication of oxidation of cyclohexyl and t-butyl groups. Based on these facts, the sixth urinary metabolite was identified as 1-[4-(2,4,5-trihydroxycyclohexoxy)phenyl]-2-2-diemthyl acetic acid.

Reference 3

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 1988 to 15 January 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

yes

Remarks:

(proposed group mean total percent recoveries were lower than the excreted percent of the dose)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

yes

Remarks:

(proposed group mean total percent recoveries were lower than the excreted percent of the dose)

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories of Kingston, NY and Portage, MA
- Age at study initiation: 5-6 weeks (preconditioned oral gavage low dose treatment); 7-8 weeks (low and high dose treatments)
- Weight at study initiation: 204-222 g (male), 192-201 g (female) - low dose group; 234-248 g (male), 187-211 g (female) - preconditioned low dose group; 214-232 g (male), 195-208 g (female) - high dose group
- Fasting period before study: approximately 12 hours prior to dosing
- Housing: polycarbonate metabolism cages
- Individual metabolism cages: yes
- Diet: Certified Purina Rodent Chow ® ad libitum
- Water: municipal water supply ad libitum
- Acclimation period: at least seven days

ENVIRONMENTAL CONDITIONS
- Temperature: 69-77 ºF
- Humidity: 43-68 %
- Air changes: minimum of 10 changes per hour
- Photoperiod: 12 hours light/12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Radiolabelled solutions were prepared by dispensing the required volume of test material into a silanised glass vial and the acetonitrile evaporated off using an air-stream of nitrogen. The desired quantity of non-labelled test material was added to the vial. A known quantity of corn oil was added and stirred into solution. All dosing solutions were prepared and dispensed into amber glass containers fitted with Teflon®-lined screw caps.

The non-labelled test material was prepared by weighing the required quantity of test material into the dosing container and adding sufficient volume of corn oil to achieve the target concentration.

Duration and frequency of treatment / exposure:

Animals were either administered a single oral dose at 25 or 200 mg/kg bw or a single daily oral gavage dose for 15 days at 25 mg/kg bw.

Dose / conc.:

25 other: mg/kg bw (single oral dose)

Dose / conc.:

200 other: mg/kg bw (single oral dose)

Dose / conc.:

25 other: mg/kg bw (daily oral dose)

No. of animals per sex per dose / concentration:

Six

Control animals:

yes

Details on study design:

Group B: Six animals per sex were administered a single oral dose (25 mg/kg) by gavage of 14C-test material.

Group C: Fifteen animals per sex were administered a single daily oral gavage dose (25 mg/kg) for 14 days of non-labelled test material and on day 15, six animals per sex were administered a single oral dose (25 mg/kg) of 14C-Omite. Three animals per sex pretreated with non-labelled test material were not treated in day 15 with 14C-test material and served as controls.

Group D: Six animals per sex were administered a single oral dose (200 mg/kg) by gavage of 14C-test material.

Group P: In a preliminary study, one male and one female were administered 25 mg/kg 14C-test material to determine the extent of elimination of the test material and to determine the appropriate duration of the sample collection period for the main study (until >90% of the actual administered dose was excreted or for 7 days, which ever occurred first).

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, cage rinse
- Time and frequency of sampling: 6, 24, 36, 48, 72, 96, 120 and 144 and 168 hours in the preliminary study; 6, 24, 36, 48, 72 and 96 hours in the main study

- Tissues and body fluids sampled: cardiac blood, liver, kidneys, skeletal muscle (biceps, femoris), perirenal fat, lungs, uterus, heat, bone, gonads, spleen, skin, brain, gastrointestinal tract and contents and residual carcass
- Time and frequency of sampling: 96 hours (after sacrifice in the main study)

Statistics:

Individual data were electronically extracted and reduced to group mean data using a SAS program.

Preliminary studies:

In the preliminary study, it was found that >95 % of the administered dose was recovered in faeces and urine within 72 hours of dosing. The duration for the main study was therefore set at 96 hours.

Details on distribution in tissues:

Concentrations of 14C-test material equivalents detected in tissues were usually below or just slightly above the limit of detection. Radioactivity in all tissues only accounted for 0.7-1.5 % of administered dose for both sexes and in all dose groups. Most of this was accounted for by the presence of test material equivalents in the liver, kidney, G.I. tract and residual carcass.

Details on excretion:

Peak urinary excretion of 14C-test material equivalents was observed at 6-24 and 6-36 hours for the low and high dose groups respectively. Urinary excretion accounted for 61.1 and 49.8 % of the total administered dose in low dose males and females respectively and 30.3 and 34.4 % in high-dose males and females respectively. In the preconditioned low dose group, peak urinary excretion occurred in between 6 and 24 hours for dosing of both sexes. Urinary excretion accounted for 53.1 and 39.4 % of the administered dose in preconditioned males and females respectively (see Table 1).

Peak faecal excretion of 14C-test material equivalents was observed at 6-24 hours in both low and high dose groups. Faecal excretion accounted for 51.3 and 61.2 % of the total administered dose in low dose males and females respectively and 74.5 and 69.9 % in high-dose males and females respectively. In the preconditioned low dose group, peak faecal excretion occurred in between 6 and 24 hours for dosing of both sexes. Faecal excretion accounted for 63.3 and 71.7 % of the administered dose in preconditioned males and females respectively (see Table 2).

Metabolites identified:

not specified

Clinical signs

There were no abnormal behavioural or overt clinical signs of toxicity exhibited in any of the animals that could be directly attributed to test material exposure. Several animals exhibited soft faeces or diarrhoea approximately 12 hours after dosing. This finding was attributed to the corn oil vehicle. Approximately 24 hours after dosing, several of the high dose group males and females exhibited hunched posture, rough fur coat and decreased activity. In addition, two females exhibited unusually aggressive behaviour.

Excretion

Table 1: Mean urinary excretion of radioactivity (% of administered dose)

Collection interval (hours)	25 mg/kg bw	200 mg/kg bw	25 mg/kg bw (preconditioned)
Males
Predose	BDL	BDL	BDL
0-6	10.7	3.8	11.4
6-24	44.2	12.9	36.2
24-36	8.2	9.1	3.8
36-48	1.2	3.0	0.9
48-72	0.8	1.2	0.5
72-96	0.3	0.4	0.4
Total	61.1	30.3	53.1
Females
Predose	BDL	BDL	BDL
0-6	12.5	8.0	8.2
6-24	30.0	15.1	27.7
24-36	5.7	7.6	2.3
36-48	0.8	2.5	0.5
48-72	0.5	0.8	0.3
72-96	0.3	0.3	0.4
Total	49.8	34.4	39.4

BDL = Below Detection Limit

Table 2: Mean faecal excretion of radioactivity (% of administered dose)

Collection interval (hours)	25 mg/kg bw	200 mg/kg bw	25 mg/kg bw (preconditioned)
Males
Predose	BDL	BDL	BDL
0-6	0	0.2	0
6-24	33.3	51.6	44.9
24-36	13.2	14.2	12.6
36-48	2.7	5.0	3.5
48-72	1.7	3.1	1.9
72-96	0.4	0.6	0.4
Total	51.3	74.5	63.3
Females
Predose	BDL	BDL	BDL
0-6	0.4	14.1	3.0
6-24	36.4	41.9	52.4
24-36	19.7	11.4	12.6
36-48	2.4	6.7	3.5
48-72	2.0	3.8	1.6
72-96	0.5	0.5	0.3
Total	61.2	69.9	71.7

BDL = Below Detection Limit

Recoveries

Group mean percent recoveries were 113.8 % (males) and 112.5 % (females) in the low dose group, 105.9 % (males) and 105.0 % (females) in the high dose group and 117.7 % (males) and 117.7 % (females) in the preconditioned low dose group.

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Urinary excretion accounted for 61.1 % (males) and 49.8 % (females) of the administered dose in the low dose group, 30.3 % (males) and 34.4 % (females) in the high dose group and 53.1 % (males) and 39.4 % (females) in the preconditioned low dose group. These findings indicate that, at the low dose level, the significance of urinary excretion as a pathway of elimination is sex-dependent. Urinary excretion also appears to be dose dependent. The decreased quantity of administered dose excreted in the urine of the high dose animals and the extended time to peak excretion (6-36 hours compared to 6-24 hours) suggests that urinary excretion pathways can become saturated at high doses. This is borne out by the increased faecal excretion seen at the high dose. The extent of urinary excretion in the preconditioned low dose group is slightly lower than that seen in the non-preconditioned low dose group, indicating that repeated exposure slightly decreases the extent and rate of urinary excretion. Based on quantity of 14C-test material equivalents recovered in urine over time, it can be concluded that a significant proportion of the administered dose was absorbed, but that this was also rapidly cleared from the body (>95 % within 0-36 hours). Faecal excretion accounted for 51.3 % (males) and 61.2 % (females) of the administered dose in the low dose group, 74.5 % (males) and 69.9 % (females) in the high dose group and 63.3 % (males) and 71.7 % (females) in the preconditioned low dose group, indicating that a large proportion of the administered dose passes through the gastrointestinal tract unabsorbed. The peak time of 6-24 hours most likely represents the transit time for the non-absorbed material to pass through the gastrointestinal tract. The increased faecal excretion at the high dose along with the decreased urinary excretion, supports the inference that urinary excretion as a pathway of elimination can become saturated and direct elimination of non-absorbed test material equivalents via the G.I. tract increases as a consequence, although elimination of some absorbed material by biliary excretion may contribute to the overall increased faecal excretion that is observed. The extent of faecal excretion in the preconditioned low dose group is slightly greater than that seen in the non-preconditioned group, indicating that repeated exposure slightly increases the extent and rate of faecal excretion (converse to the pattern of urinary excretion in the preconditioned group and further supporting the increased importance of faecal excretion as urinary pathways become saturated). The very low levels of 14C-test material equivalents detected in tissues indicate that distribution and accumulation in tissues is negligible.

Executive summary:

Rats were dosed with ¹⁴C-test material by a single oral gavage according to the following regimes: (i) 25 mg/kg bw, (ii) 25 mg/kg bw following 14 days preconditioning with 25 mg/kg non-radiolabelled test material and (iii) 200 mg/kg bw. After 96 hours, the animals were sacrificed and the total radiolabel excreted and retained in the tissues were determined (a preliminary study demonstrated that 95 % of radioactivity excreted in the urine and faeces was recovered within 96 hours of dosing).

¹⁴C-test material equivalents excreted in the urine by the low dose group accounted for 61.1 ± 4.9 % (males) and 49.8 ± 4.7 % (females) of actual administered dose. The high dose group accounted for 30.3 ± 3.0 % (males) and 34.4 ± 8.3 % (females) and the preconditioned low dose group accounted for 53.1 ± 5.2 % (males) and 39.4 ± 12.4 % (females) of actual administered dose. Peak urinary excretion was observed between 6 and 24 hours for the low and preconditioned dose groups and 6 to 36 hours for the high dose group.

¹⁴C-test material equivalents excreted in the faeces by the low dose group accounted for 51.3 ± 9.1 % (males) and 61.2 ± 8.2 % (females) of actual administered dose. The high dose group accounted for 74.5 ± 8.3 % (males) and 69.9 ± 12.5 % (females) and the preconditioned low dose group accounted for 69.3 ± 4.5 % (males) and 71.7 ± 7.84 % (females) of actual administered dose. Peak faeces excretion was observed between 6 and 24 hours for all treatment groups.

¹⁴C-test material equivalents in the tissues were generally below detection limits and accounted for approximately 1.6 % or less of the actual administered dose for all treatment groups. Measurable levels of radioactivity were generally limited to the liver, kidney, gastrointestinal tract and contents and residual carcass.

The group mean total percent recoveries for the male and female low dose group were 113.8 ± 6.3 and 112.5 ± 5.2 %, respectively, for the male and female high dose group were 105.9 ± 10.0 and 105.0 ± 9.3 %, respectively and for the male and female preconditioned group were 117.7 ± 2.9 and 111.8 ± 8.3 %, respectively.

Absorption of ¹⁴C-test material equivalents following an oral administration was estimated to be approximately 30 to 60 % of the actual administered dose (based on total ¹⁴C-radioactivity recovered in the urine and not corrected for any possible biliary excretion). Distribution and accumulation of ¹⁴C-test material equivalents in the tissues were negligible (approximately 1.5 % or less of the administered dose) at the low and high dose levels and after repeated exposure. Temporal elimination patterns of ¹⁴C-test material equivalents in the urine indicated that ¹⁴C-test material equivalents were rapidly cleared from the body i.e. approximately 95 % of the total ¹⁴C-radioactivity excreted in the urine was recovered within 0 to 36 hours after dosing for the low and preconditioned dose groups and within 0 to 48 hours for the high dose group. An estimated half-life value for elimination of ¹⁴C-test material equivalents was approximately 12 to 24 hours. A major route of elimination was by urinary excretion. Urinary excretion appeared to be dose, sex and treatment dependent. Elimination by the urinary route of excretion appeared to be saturated for the high and preconditioned dose groups and there was some indirect evidence to suggest that the biliary route of elimination supplemented excretion for these two groups. For the low and preconditioned dose groups, the extent of urinary excretion was greater for males than females.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21st January 1991 to 21st February 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

excretion

metabolism

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

yes

Remarks:

(residue levels were neither measured in caracss nor in bile, and thus only an approximated oral absorption can be determined)

GLP compliance:

yes

Radiolabelling:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Age at study initiation: 5-7 weeks
- Weight at study initiation: 22.4-25.1 g (male); 22.1-24.0 g (female)
- Fasting period before study: approximately 4 hours
- Housing: 3.2 L polycarbonate metabolism cages
- Individual metabolism cages: no
- Diet: Certified Purina Mouse Chow® ad libitum
- Water: municipal water ad libitum
- Acclimation period: approximately 3 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72-74 ºF
- Humidity: 41-43 %
- Air changes: a minimum of ten per hour
- Photoperiod: 12 hours light/12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The labelled stock solution was added into a silanised glass vial and the acetonitrile evaporated off using an air-stream of nitrogen. A desired quantity of non-labelled test material was added and a known quantity of corn oil and the solution stirred.

Duration and frequency of treatment / exposure:

Once

Dose / conc.:

150 other: mg/kg bw

No. of animals per sex per dose / concentration:

Ten

Control animals:

no

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, cage washes,
- Time and frequency of sampling: prior to dosing and at the conclusion of the following intervals: 0 to 24, 24-48, 48-72, 72-96, 96-120, 120-144 and 144-168 hours after dosing

Statistics:

Individual raw data were reduced to a group mean and standard deviation.

Details on absorption:

For male and female mice, 100.3 and 100.0 % of administered radioactivity was recovered in the urine and faeces, respectively, indicating good recovery and suggesting that absorption and retention in the tissues was negligible.

Details on excretion:

Peak urinary excretion was observed at 0-24 hours for both males and females. Urinary excretion accounted for 58.8 and 47.1 % of the total administered dose in males and females, respectively. Approximately 93 % of urinary radioactivity was recovered within 48 hours of dosing. Peak faecal excretion was also observed at 0-24 hours for male and female mice. Faecal excretion accounted for 41.5 and 52.9 % of the total administered dose in males and females, respectively. Approximately 97 and 98 % of faecal radioactivity was recovered within 72 hours of dosing for males and females, respectively. 14C-test material equivalents were all below the detection limit in urine and faecal samples taken prior to dosing (see Table 1).

Metabolites identified:

no

Table 1: Excretion of radioactivity in urine and faeces

Collection interval	% of administered dose (males)	% of administered dose (females)
Urine (plus cage rinse)
Pre-dose	BDL	BDL
0-24	42.0	33.2
24-48	12.5	10.5
48-72	3.1	2.3
72-96	0.7	0.6
96-120	0.3	0.3
120-144	0.1	0.1
144-168	0.1	0.1
Total	58.8	47.1
Faeces
Pre-dose	BDL	BDL
0-24	28.9	42.6
24-48	7.9	7.0
48-72	3.6	2.3
72-96	0.7	0.6
96-120	0.2	0.2
120-144	0.1	0.1
144-168	0.1	0.1
Total	41.5	52.9

Clinical signs

Over the test period, there were no abnormal behavioural or overt signs of toxicity that could be attributed to the test material. This was verified by body weights taken at start and end of study; all animals gained weight normally.

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Urinary excretion accounted for 58.8 and 47.1 % of the total administered dose in males and females, respectively, indicating that the extent of elimination in urine may be sex dependent. These data also indicate that a significant quantity of the administered dose was absorbed. However, the rapid urinary excretion (peak at 24 hours, 93 % of total urinary radioactivity recovered by 48 hours) indicates that the test material and its metabolites are also rapidly eliminated from the body once absorbed. This is also borne out by the total urinary plus faecal recovery of around 100 %. Faecal excretion accounted for 41.5 % (males) and 52.9 % (females) of the total administered dose, indicating that a large proportion of the administered dose was not absorbed and/or that some of the absorbed dose contributed to the radioactivity measured in the faeces by way of biliary excretion. Peak faecal excretion was also at 24 hours, with 97-98 % of faecal radioactivity recovered within 72 hours of dosing.

Executive summary:

Ten CD-1 mice per sex were dosed with ¹⁴C-test material by a single oral gavage dose of 150 mg/kg (250 µCi/kg). Serial urine and faeces specimes were collected for 168 hours after dosing. After collection of excrta, the animal metabolism cages were rinsed with methanol and distilled water. Urine, cage rinse and faeces specimens were pooled by specimen type, sex and collection period. All samples were analysed for ¹⁴C-test material equivalents by combustion techniques and/or LSC.

¹⁴C-test material equivalents excreted in the urine (including cage rinse) accounted for 58.8 % of the actual administered dose in male mice and 47.1 % in females. Peak urinary excretion of radioactivity was observed between 0 and 24 hours for both male and female mice.

¹⁴C-test material equivalents excreted in the faeces accounted for 41.5 % of the actual administered dose in male mice and 52.9 % in female mice. Peak feaces excretion of radioactivity was observed between 0 and 24 hours for both male and female mice.

For the male mice, 100.3 % of the actual administered dose was excreted in urine and faeces. For the female mice, 100 % of actual administered dose was excreted in the urine and faeces in 168 hours.

Based on total percent recovery of ¹⁴C-test material equivalents in the urine, a significant amount of the oral dose of ¹⁴C-test material was absorbed in male and female mice. Urinary excretion was a major pathway of elimination for ¹⁴C-test material equivalents. The fact that urinary excretion of ¹⁴C-test material equivalents is higher in male than female mice suggests that this elimination pathway may be sex-dependent. Temporal elimination patterns of ¹⁴C-test material equivalents indicated that the test material was eliminated rapidly from the body as approximately 93 % of the total ¹⁴C-test material equivalents in the urine was recovered in male and female mice within 0 to 48 hours after dosing. A large percentage of the dose was either not absorbed or eliminated in the faeces by biliary excretion based upon the ¹⁴C-test material equivalents recovered in the faeces. Although animal tissues and carcasses were not examined for ¹⁴C-test material-derived radioactivity, the total percent excretion of the ¹⁴C-test material equivalents in the urine and faeces of male (100.3 %) and female (100 %) mice would indicate that low levels of radioacitivity would be measured in the tissues and carcasses of the individual animals after 168 hours.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23rd June 1993 to 21st October 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

absorption

excretion

metabolism

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

14C-test material was administered by oral gavage to groups of male and female rats and mice. Blood samples were taken from the animals' retro-orbital sinus prior to treatment and 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours following oral administration. To more fully characterise plasma pharmacokinetics, the test material was administered to additional animals by the intravenous route and blood samples were obtained prior to treatment and 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours following treatment. Sufficient animals were used to provide five replicates at each time point with no more than three samples taken per animal.

GLP compliance:

yes

Radiolabelling:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories

Route of administration:

other: oral: gavage and intravenous

Vehicle:

corn oil

Duration and frequency of treatment / exposure:

Once

Dose / conc.:

150 other: mg/kg bw (single oral)

Dose / conc.:

20 other: mg/kg bw (i.v.)

Control animals:

no

Statistics:

Pharmacokinetic parameters were calculated by standard methods and PCNONLIN Nonlinear Estimation Program was used to fit observed results to pharmacokinetic models and to calculate pharmacokinetic parameters.

Plasma concentration-time profiles following oral administration best fit a one compartment pharmacokinetic model with first order input and output for both species and sexes. Following, i.v. administration, plasma concentration-time profiles best fit a two compartment open model with first oder elimination for both species and sexes.

Details on absorption:

Plasma concentration-time profiles indicated that the absorption rate was similar for males and females.

Absolute bioavailability was 73.5 % (males) and 75.5 % (females).

Details on excretion:

Total biliary elimination was 14.8 % of applied radioactivity.

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 31 minutes (males)

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 23 minutes (females)

Test no.:

#1

Toxicokinetic parameters:

Cmax: 14.3 µg/mL (males)

Test no.:

#1

Toxicokinetic parameters:

Cmax: 11.7 µg/mL (females)

Test no.:

#1

Toxicokinetic parameters:

Tmax: 2.2 ± 0.3 hours (males)

Test no.:

#1

Toxicokinetic parameters:

Tmax: 1.85 ± 0.38 hours (females)

Test no.:

#1

Toxicokinetic parameters:

other: Clearance: 15.2 mL/min/kg (males)

Test no.:

#1

Toxicokinetic parameters:

other: Clearance: 12.3 mL/min/kg (females)

Test no.:

#1

Toxicokinetic parameters:

other: Elimination half-life: 7.91 ± 1.04 hours (males)

Test no.:

#1

Toxicokinetic parameters:

other: Elimination half-life: 9.44 ± 1.42 hours (females)

Test no.:

#2

Toxicokinetic parameters:

Cmax: 713 ± 313 µg/g

Test no.:

#2

Toxicokinetic parameters:

other: Elimination half-life: 9.23 ± 2.82 hours

Test no.:

#2

Toxicokinetic parameters:

other: Clearance: 0.0.14 ± 0.003 hours

Test no.:

#2

Toxicokinetic parameters:

AUC: 11640 ± 2405 µg/g/hr

Metabolites identified:

yes

Details on metabolites:

Six major radioactive peaks were identified in bile with the relative abundance of the more polar metabolites increasing with time after dose. The first three metabolites are the same as seen in the urinary profiles and are 1-4[-(2,x-dihydroxycyclohexoxoy)phenyl]-2,2-diemthyl acetic acid (Carboxy-TBPC-diol), 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,4,5-cyclohexane (HOMe-TBPC-triol) and 1-[4-(1,1-dimehtyl-2-hydroxyethyl)phenoxy-2,x-cyclohexane diol (HOMe-TBPC-diol). A further two metabolites eluted after these three were identified as different hydroxy-cyclohexyl isomers of 1-[4-(1,1-dimethyl)phenoxy]-2-cyclohexanol (TBPC). Finally the sixth metabolite was identified as hydroxymethyl-TBPC (HOMe-TBPC). No parent compound was identified in the bile indicating rapid metabolism to more polar, readily extractable compounds.

In plasma, four major metabolites were identified, hydroxymethyl-TBPC (HOMe-TBPC), 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,x-cyclohexane diol (HOMe-TBPC-diol) and a hydroxy-cyclohexyl isomer of TBPC. Small quantities of unchanged parent compound were detected in plasma (typically <4% of administered radioactivity).

No treatment-related signs of toxicity were observed. The deaths of seven (oral) and four (i.v) animals was attributed to stress related to blood collection.

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Plasma concentration-time profiles indicate that absorption, metabolism and elimination of the test material is much more rapid in mice than rats, although HPLC radiochromatograms of bile and plasma indicate that the metabolite profiles are qualitatively the same for both species. Although there were some species-related differences in the rate of biliary excretion, the fact that total biliary excretion was the same for mice and rats (around 15-16 % of administered dose) indicates that there is no difference in the importance of biliary excretion as route of elimination in mice and rats, thereby ruling out increased biliary excretion and/or enterohepatic recirculation as a cause for the difference in chronic toxicity seen between these species.

Executive summary:

¹⁴C-test material was administered by oral gavage to groups of male and female rats and mice. Blood samples were taken from the animals' retro-orbital sinus prior to treatment and 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours following oral administration. To more fully characterise plasma pharmacokinetics, the test material was administered to additional animals by the intravenous route and blood samples were obtained prior to treatment and 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours following treatment. Sufficient animals were used to provide five replicates at each time point with no more than two samples taken per animal.

¹⁴C-test material was administered orally at a dose of 150 mg/kg bw and bile was then collected prior to treatment and 1, 2, 4, 8, 12, 24, 36 and 48 hours following administration. During bile collection, sodium taurocholate was infused as a replacement of bile salt.

Total radioactive residues were determined in plasma and bile by direct LSC.

The most significant finding was that oral absorption was 5 to 7 times faster in mice than in rats. Time to peak plasma concentration was somewhat faster for mice than rats and clearance was two-fold greater in mice. Absolute bioavailability, area under the concentration-time curve, calculated and other observed peak plasma levels and elimination parameters were the same for both species. These pharmacokinetic parameters revealed a more prolonged plateau in the plasma concentration-time curve for rats than for mice suggesting a species difference in enterohepatic recirculation.

¹⁴C-test material was well absorbed, and the volumes of distribution, which approximated total body water volumes for both species, indicated distribution throughout the body. The most striking finding was that elimination rate was slower following i.v. administration suggesting a third compartment, perhaps a reservoir. Such a reservoir might be a consequence of high levels of ¹⁴C-test material in the systemic circulation only after i.v. administration.

No significant sex differences were observed in the bile pharmacokinetic study. Significantly greater areas under the bile concentration-time curve, higher peak biliary concentration of radioactivity and shorter elimination half-life for mice than for rats were observed. While these pharmacokinetic parameters were significantly different, total biliary elimination was similar for rats and mice. There was no evidence to support major differences in enteroheptaic mechanisms between the species.

There was no qualitative differences in the metabolite profiles of pooled samples from female rats and mice 24 hours following administration of ¹⁴C-test material. Profiles confirmed a similar qualitative metabolite profiles in bile samples from male rats and mice. No species difference were revealed in the HPLC profiles of rat and mouse plasma.

Reference 6

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7th July 1993 to 6th September 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

absorption

excretion

metabolism

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

14C-test material was administered by oral gavage to groups of male and female rats and mice. Blood samples were taken from the animals' retro-orbital sinus prior to treatment and 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours following oral administration. To more fully characterise plasma pharmacokinetics, the test material was administered to additional animals by the intravenous route and blood samples were obtained prior to treatment and 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours following treatment. Sufficient animals were used to provide five replicate at each time point with no more than three samples taken per animal.

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories

Route of administration:

other: oral: gavage and intravenous

Vehicle:

corn oil

Duration and frequency of treatment / exposure:

Once

Dose / conc.:

150 other: mg/kg bw (single oral)

Dose / conc.:

20 other: mg/kg bw (i.v.)

Control animals:

no

Statistics:

Pharmacokinetic parameters were calculated by standard methods and PCNONLIN Nonlinear Estimation Program was used to fit observed results to pharmacokinetic models and to calculate pharmacokinetic parameters.

Plasma concentration-time profiles following oral administration best fit a one compartment pharmacokinetic model with first order input and output for both species and sexes. Following, i.v. administration, plasma concentration-time profiles best fit a two compartment open model with first oder elimination for both species and sexes.

Details on absorption:

Plasma concentration-time profiles indicated that the absorption rate was similar for males and females.

Absolute bioavailability was 78.7 % for males and 79.7 % for females.

Details on excretion:

Ttal biliary elimination was 15.5 % of applied radioactivity.

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 159 minutes (males)

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 154 minutes (females)

Test no.:

#1

Toxicokinetic parameters:

Cmax: 11.4 µg/mL (males)

Test no.:

#1

Toxicokinetic parameters:

Cmax: 9.34 µg/mL (females)

Test no.:

#1

Toxicokinetic parameters:

Tmax: 6.9 hours (males)

Test no.:

#1

Toxicokinetic parameters:

Tmax: 7.1 hours (females)

Test no.:

#1

Toxicokinetic parameters:

other: Clearance: 8.44 mL/min/kg (males)

Test no.:

#1

Toxicokinetic parameters:

other: Clearance: 9.46 mL/min/kg (females)

Test no.:

#1

Toxicokinetic parameters:

other: Elimination half-life: 10.2 ± 1.3 hours (males)

Test no.:

#1

Toxicokinetic parameters:

other: Elimination half-life: 11.3 ± 1.6 hours (females)

Test no.:

#2

Toxicokinetic parameters:

Cmax: 326 ± 128 µg/g

Test no.:

#2

Toxicokinetic parameters:

other: Elimination half-life: 21.4 ± 12.0 hours

Test no.:

#2

Toxicokinetic parameters:

other: Clearance: 0.020 ± 0.008 hours

Test no.:

#2

Toxicokinetic parameters:

AUC: 8836 ± 3777 µg/g/hr

Metabolites identified:

yes

Details on metabolites:

Six major radioactive peaks were identified in bile with the relative abundance of the more polar metabolites increasing with time after dose. The first three metabolites are the same as seen in the urinary profiles and are 1-4[-(2,x-dihydroxycyclohexoxoy)phenyl]-2,2-diemthyl acetic acid (Carboxy-TBPC-diol), 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,4,5-cyclohexane (HOMe-TBPC-triol) and 1-[4-(1,1-dimehtyl-2-hydroxyethyl)phenoxy-2,x-cyclohexane diol (HOMe-TBPC-diol). A further two metabolites eluted after these three were identified as different hydroxy-cyclohexyl isomers of 1-[4-(1,1-dimethyl)phenoxy]-2-cyclohexanol (TBPC). Finally the sixth metabolite was identified as hydroxymethyl-TBPC (HOMe-TBPC). No parent compound was identified in the bile indicating rapid metabolism to more polar, readily extractable compounds.

In plasma, four major metabolites were identified, hydroxymethyl-TBPC (HOMe-TBPC), 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,x-cyclohexane diol (HOMe-TBPC-diol) and a hydroxy-cyclohexyl isomer of TBPC. Small quantities of unchanged parent compound were detected in plasma (typically <4 % of administered radioactivity).

No treatment-related signs of toxicity were observed.

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Plasma concentration-time profiles indicate that absorption, metabolism and elimination of the test material is much more rapid in mice than rats, although HPLC radiochromatograms of bile and plasma indicate that the metabolite profiles are qualitatively the same for both species. Although there were some species-related differences in the rate of biliary excretion, the fact that total biliary excretion was the same for mice and rats (around 15-16 % of administered dose) indicates that there is no difference in the importance of biliary excretion as route of elimination in mice and rats, thereby ruling out increased biliary excretion and/or enterohepatic recirculation as a cause for the difference in chronic toxicity seen between these species.

Executive summary:

¹⁴C-test material was administered by oral gavage to groups of male and female rats and mice. Blood samples were taken from the animals' retro-orbital sinus prior to treatment and 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours following oral administration. To more fully characterise plasma pharmacokinetics, the test material was administered to additional animals by the intravenous route and blood samples were obtained prior to treatment and 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 hours following treatment. Sufficient animals were used to provide five replicate at each time point with no more than three samples taken per animal.

¹⁴C-test material was administered orally at a dose of 150 mg/kg bw and bile was then collected prior to treatment and 1, 2, 4, 8, 12, 24, 36 and 48 hours following administration. During bile collection, sodium taurocholate was infused as a replacement of bile salt.

Total radioactive residues were determined in plasma and bile by direct LSC.

The most significant finding was that oral absorption was 5 to 7 times faster in mice than in rats. Time to peak plasma concentration was somewhat faster for mice than rats and clearance was two-fold greater in mice. Absolute bioavailability, area under the concentration-time curve, calculated and other observed peak plasma levels and elimination parameters were the same for both species. These pharmacokinetic parameters revealed a more prolonged plateau in the plasma concentration-time curve for rats than for mice suggesting a species difference in enterohepatic recirculation.

¹⁴C-test material was well absorbed, and the volumes of distribution, which approximated total body water volumes for both species, indicated distribution throughout the body. The most striking finding was that elimination rate was slower following i.v. administration suggesting a third compartment, perhaps a reservoir. Such a reservoir might be a consequence of high levels of ¹⁴C-test material in the systemic circulation only after i.v. administration.

No significant sex differences were observed in the bile pharmacokinetic study. Significantly greater areas under the bile concentration-time curve, higher peak biliary concentration of radioactivity and shorter elimination half-life for mice than for rats were observed. While these pharmacokinetic parameters were significantly different, total biliary elimination was similar for rats and mice. There was no evidence to support major differences in enterohepatic mechanisms between the species.

There was no qualitative differences in the metabolite profiles of pooled samples from female rats and mice 24 hours following administration of ¹⁴C-test material. Profiles confirmed a similar qualitative metabolite profiles in bile samples from male rats and mice. No species difference were revealed in the HPLC profiles of rat and mouse plasma.

Reference 7

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th June 1998 to 22nd July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: approximately 8 weeks old
- Fasting period before study: 16-18 hours
- Housing: Nagalene® metabolism cages
- Individual metabolism cages: yes
- Diet: Certified Rodent Chow® Number 5002 ad libitum
- Water: tap water ad libitum
- Acclimation period: 24 hours prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 18-26 ºC
- Humidity: 30-70 %
- Air changes: equal to ten or more
- Photoperiod: 12 hours light/12 hours dark

IN-LIFE DATES: From: 18th June 1998 To: 25th June 1998

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
15 mL corn oil added to test material stock solution reduce to dryness under nitrogen stream. Prior to dosing, the dose solution was stirred for 30 minutes.

VEHICLE
- Concentration in vehicle: 5 mL/kg

Duration and frequency of treatment / exposure:

Once

Dose / conc.:

150 other: mg/kg bw

No. of animals per sex per dose / concentration:

Five

Control animals:

yes, concurrent vehicle

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, cage washes
- Time and frequency of sampling: 24 hour intervals over a 96 hour period

Statistics:

Statistical comparisons were conducted using the two-sample t-test.

Details on distribution in tissues:

2.61 % of administered dose was detected in rat carcasses.

Details on excretion:

Peak urinary excretion was observed at 0-24 hours with 75 % of urinary excretion by 24 hours and 96 % by 48 hours. Urinary excretion accounted for 24.9 % of the total administered dose. Peak faecal excretion was also observed at 0-24 hours with 80-88 % of faecal excretion by 24 hours and 96-98 % by 48 hours. Faecal excretion accounted for 48.1 % of the total administered dose. The total percentage of radioactivity in the cage wash at 96 hours was about 5.7 % of administered dose (see Table 1).

Metabolites identified:

no

Throughout the test period, all animals appeared clinically normal. On necropsy, there were no apparent gross lesions on any of the internal organs.

Total recovery of radioactivity amounted to 81.27 %, respectively. The <100 % recovery was ascribed to volatile metabolites in exhaled air and/or incomplete collection for excreta.

Table 1: Excretion of radioactivity

Collection interval (hours)	% of administered dose
Urine
24	18.83
48	5.10
72	0.54
96	0.39
Total	24.86
Faeces
24	42.17
48	4.75
72	0.81
96	0.37
Total	48.10
Cage wash
24	4.39
48	0.67
72	0.11
96	0.54
Total	5.71

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Urinary excretion accounted for 24.9 % of the total administered dose, while faecal excretion accounted for 48.1 %. The greater importance of urinary excretion in mice indicates that mice absorb a significantly greater quantity of propargite than rats, whereas the greater importance of faecal excretion in rats indicates that a larger proportion of the administered dose is not absorbed than is the case for mice. However, urinary excretion was equally rapid in both species (peak at 24 hours, near complete at 48 hours), indicating that the test material and its metabolites are rapidly eliminated from the body once absorbed. Faecal excretion was also equally rapid in both species.

Executive summary:

Five male rats and 40 male mice received a single oral dose of 150 mg [¹³C/¹⁴C]test material/kg bw in a volume of 5 mL/kg. Mice were housed in pools of 5 mice per cage. Urine and faeces were collected at 24 hour intervals from dosing until termination at 96 hours post-dose. A cage wash was conducted immediately following each of the collections. A brief necropsy was conducted at termination and the major internal organs examined. Carcasses were retained for analysis.

Throughout the study all animals appeared clinically normal. Upon necropsy, there were no apparent gross effects on any of the major internal organs. Rats and mice excreted the radiolabelled substance at about the same rate (as a % of administered dose) but mice excreted significantly more radiolabel in the urine than rats (39.9 % administered dose compared to 24.86 %). The rats correspondingly excreted more radiolabel in the faeces than the mice. At the 96 hour study termination, similar amounts of radiolabel were present in the carcass in terms of administered dose and ppm.

Reference 8

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th June 1998 to 22nd July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: approximately 6 weeks old
- Fasting period before study: 16-18 hours
- Housing: Nagalene® metabolism cages
- Individual metabolism cages: no
- Diet: Certified Rodent Chow® Number 5002 ad libitum
- Water: tap water ad libitum
- Acclimation period: 24 hours prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 18-26 ºC
- Humidity: 30-70 %
- Air changes: equal to ten or more
- Photoperiod: 12 hours light/12 hours dark

IN-LIFE DATES: From: 18th June 1998 To: 25th June 1998

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
15 mL corn oil added to test material stock solution reduce to dryness under nitrogen stream. Prior to dosing, the dose solution was stirred for 30 minutes.

VEHICLE
- Concentration in vehicle: 5 mL/kg

Duration and frequency of treatment / exposure:

Once

Dose / conc.:

150 other: mg/kg bw

No. of animals per sex per dose / concentration:

40

Control animals:

yes, concurrent vehicle

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, cage washes
- Time and frequency of sampling: 24 hour intervals over a 96 hour period

Statistics:

Statistical comparisons were conducted using the two-sample t-test.

Details on distribution in tissues:

2.06 % of administered dose was detected in mouse carcasses.

Details on excretion:

Peak urinary excretion was observed at 0-24 hours with 75 % of urinary excretion by 24 hours and 96 % by 48 hours. Urinary excretion accounted for 39.9 % of the total administered dose. Peak faecal excretion was also observed at 0-24 hours with 80-88 % of faecal excretion by 24 hours and 96-98 % by 48 hours. Faecal excretion accounted for 29.2 % of the total administered dose. The total percentage of radioactivity in the cage wash at 96 hours was about 5.6 % of administered dose (see Table 1).

Metabolites identified:

no

Throughout the test period, all animals appeared clinically normal. On necropsy, there were no apparent gross lesions on any of the internal organs.

Total recovery of radioactivity amounted to 76.76 %, respectively. The <100 % recovery was ascribed to volatile metabolites in exhaled air and/or incomplete collection for excreta.

Table 1: Excretion of radioactivity

Collection interval (hours)	% of administered dose
Urine
24	26.86
48	9.45
72	1.16
96	0.44
Total	39.90
Faeces
24	23.36
48	4.56
72	0.56
96	0.38
Total	29.22
Cage wash
24	3.65
48	1.22
72	0.38
96	0.33
Total	5.58

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Urinary excretion accounted for 39.9 % of the total administered dose, while faecal excretion accounted for 29.2 %. The greater importance of urinary excretion in mice indicates that mice absorb a significantly greater quantity of propargite than rats, whereas the greater importance of faecal excretion in rats indicates that a larger proportion of the administered dose is not absorbed than is the case for mice. However, urinary excretion was equally rapid in both species (peak at 24 hours, near complete at 48 hours), indicating that the test material and its metabolites are rapidly eliminated from the body once absorbed. Faecal excretion was also equally rapid in both species.

Executive summary:

Five male rats and 40 male mice received a single oral dose of 150 mg [¹³C/¹⁴C]test material/kg bw in a volume of 5 mL/kg. Mice were housed in pools of 5 mice per cage. Urine and faeces were collected at 24 hour intervals from dosing until termination at 96 hours post-dose. A cage wash was conducted immediately following each of the collections. A brief necropsy was conducted at termination and the major internal organs examined. Carcasses were retained for analysis.

Throughout the study all animals appeared clinically normal. Upon necropsy, there were no apparent gross effects on any of the major internal organs. Rats and mice excreted the radiolabelled substance at about the same rate (as a % of administered dose) but mice excreted significantly more radiolabel in the urine than rats (39.9 % administered dose compared to 24.86 %). The rats correspondingly excreted more radiolabel in the faeces than the mice. At the 96 hour study termination, similar amounts of radiolabel were present in the carcass in terms of administered dose and ppm.

Reference 9

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8th March 2002 to 1st December 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

(samples containing radioactivity below twice background were used for calculating baseline distribution and HPLC recovery)

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: 6 weeks
- Fasting period before study: 16 - 18 hours
- Housing: Nalgene metabolism cages
- Individual metabolism cages: yes
- Diet: Certified Rodent Chow No. 5002 ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 26 ºC
- Humidity: 30 - 70 %
- Air changes (per hr): 10 or more changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Propargite in corn oil was administered to animals at a dose level of 25 mg/kg bw. A volume of approximately 5 mL/kg was delivered to each animal. Each animal recieved approximately 80 - 100 µCi/kg bw.

Duration and frequency of treatment / exposure:

14 consecutive daily doses (25 mg/kg bw) of [12C]propargite followed by a single oral dose of [14C]propargite at 25 mg/kg bw on the 15th day.

Dose / conc.:

25 other: mg/kg bw

No. of animals per sex per dose / concentration:

5

Control animals:

yes, concurrent vehicle

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, cage washes, brain, heart, lungs, liver, adrenals, kidneys, GI tract (and contents), bone (femur), muscle (semimembranosus), mesentric fat, testes (ovaries), uterus, spleen , pancreas, thyroid, bone marrow, thymus.
- Time and frequency of sampling: excreta and cage washes at 24 hour intervals. Blood and tissues at termination.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces

Details on distribution in tissues:

Radiolabel was present in the tissues and G.I. tract/contents at levels of 0.56 and 0.23 % of administered dose in males and females, respectively. Of this, 17.9 % (males) and 34.8 % (females) was present in the G.I. tract/contents, while 51.8 % of the radioactivity measured in male tissues was found in the carcass (0 % in female carcass). In terms of concentrations in individual tissue types/organs, the greatest concentration of administered radioactivity was found in the adrenals (1.44 - 2.18 µg-eq/g) and the kidneys (1.02 - 1.77 µg-eq/g). Total recovery in males and females (radioactivity excreted + radioactivity in tissues) was 98.4 and 97.6 %, respectively (see Table 2).

Details on excretion:

Peak urinary excretion was observed at 0-24 hours for both males and females, with >93 % of urinary excretion occurring within 24 hours post-dose. In total, urinary excretion accounted for 59.15 and 43.58 % of the total administered dose in males and females, respectively. Peak faecal excretion was also observed at 0-24 hours for both males and females, with 81.2 % (males) and 85.9 % (females) of faecal excretion occurring within 24 hours post-dose. In total, faecal excretion accounted for 35.42 and 50.89 % of the total administered dose in males and females, respectively. Total radioactivity in cage washes amounted to 3.3 % (males) and 2.87 % (females) (see Table 1).

Metabolites identified:

yes

Details on metabolites:

Following separation by HPLC and LSC of effluents collected over 0.5 minute intervals, the reconstructed radio-chromatograms indicated the presence of ten peaks (metabolites) in male and 8 peaks in female urine, collected over 0-24 hours. In terms of relative abundance, peak 2 was the most abundant in males, accounting for 30.9 % of total radioactive residues (TRR), while peak 4 was the most abundant in female urine (26.5 % TRR). Urine collected over 24- 48 hours contained fewer metabolites (2 and 2 in males and females, respectively). Following separation of faecal extracts by HPLC and LSC of effluents collected over 0.5 minute intervals, the reconstructed radio-chromatograms indicated the presence of 6 peaks in male and 7 peaks in female faeces, collected over 0-24 hours. In terms of relative abundance, peak 6 was the most abundant in males, accounting for 29.4 % of total radioactive residues (TRR), while peak 5 was the most abundant in female faeces (35.8 % TRR). Faeces collected over 24- 48 hours contained fewer metabolites (2 and 4 in males and females, respectively) (see Tables 3-5. Note that some peaks are actually the same metabolite. In total six distinct metabolites were identified in rat excreta).

Throughout the test period, all animals appeared clinically normal with no apparent changes in behaviour. Body weight increased steadily during the dosing period. No apparent gross lesions were observed on any of the internal organs following necropsy.

Table 1: Excretion of radioactivity in urine and faeces

Collection interval (hours post-dose)	% of administered dose (males)	% of administered dose (females)
Urine
24	55.11	41.24
48	2.58	1.65
72	0.61	0.34
96	0.41	0.15
120	0.21	0.09
144	0.13	0.06
168	0.10	0.04
Total	59.15	43.58
Faeces
24	28.76	43.72
48	5.23	5.31
72	0.85	1.24
96	0.28	0.29
120	0.16	0.13
144	0.09	0.11
168	0.07	0.09

Table 2: Distribution of radioactivity in tissues

Tissue organ/type	Males (mean % administered dose)	Females (mean % administered dose)
Adrenals	0.00	0.00
Bone marrow	0.00	0.00
Pancreas	0.00	0.00
Spleen	0.00	0.00
Uterus	-	0.00
Thymus	0.00	0.00
Thyroid	0.00	0.00
Brain	0.01	0.01
Carcass	0.29	0.00
Gastrointestinal tract and contents	0.10	0.08
Heart	0.01	0.01
Kidneys	0.06	0.04
Liver	0.08	0.09
Lungs	0.00	0.00
Muscle	0.00	0.00
Fat	0.01	0.01
Testes	0.01	0.00
Bone	0.00	0.01
Blood	0.00	0.00

Table 3: Distribution of radioactivity and quantitation of ¹⁴C-components

Fraction/Sex	Peak No.	^14C-residue	% TRR	% administered dose
Urine
Males	1	HOMe-TBCP-triol sulphate	7.98	4.40
	2	Carboxy-TBPC-diol	30.9	17.0
	3	Carboxy-TBPC-diol	6.97	3.84
	4	HOMe-TBPC-triol	18.1	10.0
	5	HOMe-TBPC diol sulphate	5.75	3.17
	6	HOMe-TBPC-triol	1.97	1.09
	7	HOMe-TBPC-triol	7.41	4.08
	8	HOME-TBPC-diol sulphate	4.39	2.42
	9	HOME-TBPC-diol	9.87	5.44
	10	Carboxy-TBPC	2.97	1.64
Females	1	HOMe-TBCP-triol sulphate	12.8	5.28
	2	Carboxy-TBPC-diol	18.9	7.79
	3	HOME-TBPC-triol	10.5	4.33
	4	HOME-TBPC-diol sulphate	26.5	10.9
	5	HOMe-TBPC-diol sulphate	7.94	3.27
	6	HOME-TBPC-diol sulphate	5.28	2.18
	7	HOMe-TBPC-diol	3.62	1.49
	8	Carboxy-TBPC	8.47	3.49
Faeces
Males	1	HOMe-TBCP-triol sulphate	4.47	1.29
	2	Carboxy-TBPC-diol	28.0	8.04
	3	Carboxy-TBPC-diol	11.1	3.19
	4	HOMe-TBPC-diol sulphate	11.9	3.42
	5	HOME-TBPC-diol	2.77	0.796
	6	Propargite	29.4	8.46
	-	Post Extraction Solids (PES)	3.10	0.892
Females	1	HOMe-TBCP-triol sulphate	12.2	5.34
	2	HOME-TBPC-diol sulphate	4.04	1.77
	3	HOMe-TBPC-triol sulphate	4.44	1.94
	4	Carboxy-TBPC-diol	15.0	6.56
	5	HOMe-TBPC-diol sulphate	35.8	15.6
	6	HOMe-TBPC-diol sulphate	6.45	2.82
	7	Propargite	8.21	3.59
	-	Post Extraction Solids (PES)	1.40	0.612

Table 4: Distribution of radioactivity and quantitation of ¹⁴C-components

Fraction/Sex	Peak no.	^14C-residue	% TRR	% administered dose
Urine
Males	1	Carboxy-TBPC-diol	34.0	0.877
	2	HOMe-TBPC-triol	22.4	0.578
Females	1	Carboxy-TBPC-diol	33.1	0.546
	2	HOMe-TBPC-triol	30.2	0.499
Faeces
Males	1	Carboxy-TBPC-diol	59.5	3.11
	2	HOMe-TBPC-diol sulphate	15.5	0.812
	-	Post Extraction Solids (PES)	2.14	0.112
Females	1	HOMe-TBPC-triol sulphate	24.3	1.29
	2	Carboxy-TBPC-diol	22.9	1.22
	3	HOMe-TBPC-diol sulphate	33.6	1.78
	4	Propargite	6.49	0.345
	-	Post Extraction Solids (PES)	1.83	0.097

 

Table 5: Quantitation comparison of ¹⁴C-components

Metabolites	% administration dose (males)	% administration dose (females)
1) HOMe-TBPC-triol sulphate	5.69	13.9
2) Carboxy-TBPC-diol	36.0	16.1
3) HOMe-TBPC-triol	15.8	4.83
4) HOMe-TBPC-diol sulphate	9.82	38.4
5) HOMe-TBPC-diol	6.24	1.49
6) Carboxy-TBPC	1.64	3.49
7) Propargite	8.46	3.93

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Excretion was rapid in rats with peak urinary/faecal excretion occurring within 0-24 hours. In total, urinary excretion accounted for 59.15 and 43.58 % of total administered dose in males and females, respectively, whereas faecal excretion accounted for 35.42 and 50.89 % of the total administered dose in males and females. The males excreted significantly more radioactivity via the urine than the females, but conversely, the females excreted significantly more radioactivity via the faeces than the males. >93 % (urine) and >81 % (faeces) of total excretion was excreted by both sexes in the first 24 hours post-dose. Total excretion amounted to >97 % in both sexes.

Radiolabel was present in the tissues and G.I. tract/contents at levels of 0.56 and 0.23 % of administered dose in males and females, respectively. The majority of this was present in the G.I. tract/contents (18 % males, 35 % females) and/or the carcass (51 % males, 0 % females).

The most abundant metabolite in males was 1-[4-(2,x-dihydroxy-cyclohexoxy)pheny1]-2,2-dimethylacetic acid (Carboxy-TBPC-diol) accounting for 20.8 and 11.2 % of administered dose in urine and faeces, respectively. The most abundant metabolite in females was 1-[4-(1,1-dimethy1-2-hydroxyethyl)phenoxyl-2,x,-cyclohexane-diol sulphate (HOMe-TBPC-diol sulphate) accounting for 16.4 and 20.2 % of administered dose in urine and faeces, respectively. Other metabolites identified in rat excreta were as follows:
2-[4-(2-hydroxy-1,1-dimethylethyl)phenoxy] cyclohexane-1,x,x'-triol sulphate (HOMe-TBPC-triol sulphate)
2-[4-(2-hydroxy-1,1-dimethylethyl)phenoxy]cyclohexane-1,x,x'-triol (HOMe-TBPC-triol)
1-[4-(1,1-dimethy1-2-hydroxyethyl)phenoxy]-2,x,-cyclohexane-diol (HOMe-TBPC-diol)
3-{4-[(2-hydroxycyclohexyl)oxylphenyl}-3-methylbutanoic acid (Carboxy-TBPC)

The absence of the parent compound in rat urine and the relatively small quantity detected in faeces indicates extensive metabolism in rats. Most metabolites were found in both urine and faeces, with the exception of HOMe-TBPC-triol which was found in urine only. Although all metabolites were found in both rats, the relative proportions of the various metabolites differed between the sexes. Propargite was 2-fold greater in excreta of females than in males, while the level of sulphate conjugates was 3.5 x greater in females than in the males. In contrast, the level of Carboxy-TBPC and HOMe-TBPC-non-sulphate type metabolites was much higher in males than females.

The identified metabolites resulted from the hydrolysis of the propynyl sulphite side chain of the parent compound and subsequent oxidation of a methyl group of the tert-butyl portion and hydroxylation of the cyclohexyl moiety to yield HOMe-TBPC-diol and HOMe-TBPC-triol. Hydroxylation of the cyclohexyl moiety produced a mixture of positional and/or stereochemical (axial/equatorial) hydroxy isomers. These metabolites in turn underwent conjugation with sulphate to produce HOMe-TBPC-diol sulphate and HOMe-TBPC-triol sulphate.

Executive summary:

Rats received 14 consecutive doses of ¹²C-test material followed by a single oral dose of ¹⁴C-test material on the 15th day. Each dose was administered at a dose level of 25 mg/kg bw. Urine and faeces were collected at 24 hour intervals after administration of ¹⁴C-test material and the animals were terminated at 168 hours post-dose.

Essentially all of the radioactivity was excreted within a week of the radiolabelled dose administration. Less than 1 % of the dose remained within the body indicating that a repeat dosing at the level of 25 mg/kg bw does not affect tissue accumulation.

¹⁴C-test material equivalents excreted in the urine of males and females were approximately 59 and 44 % of actual administered dose. Peak excretion of radioactivity in urine was observed 0-24 hours following the radioactive dose. ¹⁴C-test material equivalents in the faces of males and females were approximately 35 and 51 % respectively. Peak excretion of radioactivity in faeces was observed 0-24 hours following the radioactive dose. The total percent recovery was approximately 98 % for both males and females.

The most abundant metabolite in male urine and faeces accounted for 20.8 and 11.3 % of the administered dose respectively and was identified as 1-[4-(2,x-dihydroxycyclohexoxy)-phenyl-2,2-dimethylacetic acid (Carboxy-TBPC-diol). The most abundant metabolite in female urine and faeces accounted for 16.4 and 20.2 % of the administered dose respectively and was identified as 1-[4-(1,1-dimethyl-2-hydroxyethyl)phenoxy]-2,x-cyclohexane-diol sulfate (HOMe-TBPC-diol sulfate).

The following metabolites were identified in rat excreta:

2-[4-(2 -hydroxy-1,1-dimethylethyl)phenoxy]cyclohexane-1,x,x'-triol sulfate (HOMe-TBPC-triol sulfate)

2-[4-(2 -hydroxy-1,1-dimethyl(phenoxy]cyclohexane-1,x-diol sulfate (HOMe-TBPC-diol sulfate)

3-{-4-[(2,x-dihydroxycyclohexyl)oxy]phenyl}-3-methylbutanoic acid (Carboxy-TBPC-diol)

2-[4-(2 -hydroxy-1,1-dimethylethyl)phenoxy]cyclohexane-1,x,x'-triol (HOMe-TBPC-triol)

2-[4-(2 -hydroxy-1,1-dimethylethyl)phenoxy]cyclohexane-1,x-diol (HOMe-TBPC-diol)

3-{4-[(2-hydroxycyclohexyl)oxy]phenyl}-2-methylbutanoic acid (Carboxy-TBPC)

A small quantity of the parent compound was identified in the faecal extracts.

These metabolites resulted from the hydrolysis of the propnyl sulfite side chain of the parent compound and subsequent hydroxylation of a methyl group of the tert-butyl portion and hydroxylation of the cyclohexyl moiety to yield HOMe-TBPC-diol and HOMe-TBPC-triol. Hydroxylation of the cyclohexyl moiety produced a mixture of positional and/or stereochemical (axial/equatorial) hydroxy isomers. These metabolites in turn underwent conjugation with sulfate to produce HOMe-TBPC-diol sulfate and HOMe-TBPC-triol sulfate or further oxidation of the hydroxylated tert-butyl group to produce Carboxy-TBPC and Carboxy-TBPC-diol.

Reference 10

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January 2015 to 08 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of this study was to compare the metabolism in vitro of [14C]Propargite using hepatocytes from mouse, rat (two strains, namely Sprague-Dawley and Fischer 344), dog and human. Preliminary incubations of [14C]Propargite (nominal concentrations: 1,3,10, 30 and 100 µM) were conducted with pooled male and female Sprague-Dawley rat cryopreserved hepatocytes (1 x 10^6 viable cells) for 0, 0.25, 0.5, 1 and 3 hours, each as a single sample in order to check the metabolic stability of [14C]Propargite. Supernatants from these cultures were analysed by reversed-phase HPLC. The potential cytotoxicity of [14C]Propargite was assessed by the measurement of the leakage of lactate dehydrogenase (LDH). Interspecies comparison incubations of [14C]Propargite metabolism (nominal concentration: 10 μM) using cryopreserved hepatocytes from pooled male and female mouse, rat (separately from each of Sprague-Dawley and Fischer 344), dog and human was subsequently conducted at incubation times of 0.25 and 1 hour, in a similar manner to the preliminary incubation. Samples were processed as for the preliminary incubation. In the preliminary incubations [14C]Propargite was extensively metabolised by Sprague-Dawley rat cryopreserved hepatocytes after 3 hours incubation as exemplified by the depletion of parent. Based on the results, a [14C]Propargite concentration of 10 μM and timepoints of 0.25 and 1 hour were selected for the interspecies comparison incubations. In the main interspecies comparison incubations, were conducted at a final concentration of 10 µM with pooled male and female mouse, rat (each of Sprague-Dawley and Fischer 344), dog and human cryopreserved hepatocytes for 0.25 and 1 hours.

GLP compliance:

yes

Radiolabelling:

yes

Species:

other: human, dog, rat and mouse

Strain:

other: Human: N/A; dog: beagle; rat: Sprague Dawley, Fischer 344; and mouse: CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

All cryopreserved hepatocytes were obtained from Bioreclamation IVT (formerly Celsis IVT) and delivered stored frozen in liquid nitrogen. On receipt, the hepatocytes were transferred to permanent storage in liquid nitrogen.
All hepatocytes were prepared as a pool of the male and female donors. Each vial used contained at least 1 million cells (mouse) or 5 million cells (all other species). One vial was used per sex per species. Three vials per sex were used for the Sprague-Dawley strain rat in a preliminary phase.

Route of administration:

other: in vitro

Vehicle:

other: acetonitrile

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The solubility of non-radiolabelled test material was determined at a nominal concentration of 10 mM in each of acetonitrile, ethanol and methanol by weighing out separate aliquots of non-radiolabelled test material followed by the addition of an appropriate volume of solvent to give a nominal concentration of 10 mM in each solvent. The assessment of dissolution was by visual inspection.
The aqueous solubility of each solvent soluble test material solution was then assessed by taking an aliquot (10 µL) of each of solution and spiking each into separate aliquots of Williams’ media E (990 µL) to assess solubility under aqueous conditions.
The test material appeared to be fully soluble at 10 mM in each of acetonitrile, ethanol and methanol by visual inspection, whilst in aqueous media 10 mM the test material in acetonitrile gave the best solubility; at final test material concentration of 100 µM (with 1 % (v/v) organic contribution in aqueous media). Consequently, acetonitrile was selected as the solvent of choice for the dissolution of the test material in this study.
Individual solutions of each of the test material and the reference compounds O-10, O-34, OMT-B, O-70, O-72 and O-73 were prepared at 1 mg/mL. An aliquot (100 µL) of each was then combined to give a standard mix containing 0.14 mg/mL of each reference standard which was used for LC-MS analysis.

Duration and frequency of treatment / exposure:

- Preliminary incubation: Incubation times 0, 0.25, 0.5, 1 and 3 hours
- Interspecies comparison incubations: 0.25 and 1 hour

Dose / conc.:

1 other: µM

Remarks:

Preliminary incubation

Dose / conc.:

3 other: µM

Remarks:

Preliminary incubation

Dose / conc.:

10 other: µM

Remarks:

Preliminary incubation

Dose / conc.:

30 other: µM

Remarks:

Preliminary incubation

Dose / conc.:

100 other: µM

Remarks:

Preliminary incubation

Dose / conc.:

10 other: µM

Remarks:

Interspecies comparison incubations

No. of animals per sex per dose / concentration:

Number of replicates: one

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

The positive control was [14C]7 Ethoxycoumarol at a concentration of 50 µM for 3 h in duplicate with rat hepatocytes.

Details on study design:

Doses and incubation times in the main study were selected on basis of results in the prelinary incubations. See Details on dosing and sampling

Details on dosing and sampling:

PRELIMINARY INCUBATION
Incubations of [14C]test material were conducted with cryopreserved Sprague-Dawley rat hepatocytes as follows:
- Concentrations: 0 (LDH assay only), 1, 3, 10, 30 and 100 µM
- Incubation times: 0, 0.25, 0.5, 1 and 3 hours
- Incubation temperature: 37 °C
- Number of replicates: One
- Cell concentration: 1 x 10^6 viable cells per mL of culture medium
- Volume of incubation medium: 1 mL (3.5 mL for LDH assay)

The incubation components were mixed together in polyethylene vials for each sample as follows:
- Supplemented Williams’ Medium E containing 1.01 × 10^6 viable cells and 10.1 % (v/v) foetal calf serum
- [14C]test material (10 µL of either 0.1, 0.3, 1, 3, 10 mM solution in acetonitrile or acetonitrile only (LDH solvent control))
Note: Volumes quoted were adjusted for the 3.5 mL LDH assay incubations.

Following the final addition of [14C]test material (or acetonitrile), 0 h samples were terminated immediately. For all other incubation times, each vial was placed in an orbital shaking water bath to commence the incubation (95 % O2 / 5 % CO2, 37 °C).
The following control incubations were also conducted in parallel: Incubation of [14C]test material for 3 h in the absence of hepatocytes (single incubation at 1, 3, 10, 30 and 100 µM).
Positive control samples were carried out, incubating [14C]7 EC at a concentration of 50 µM for 3 h in duplicate with rat hepatocytes
At the end of the requisite incubation period, each sample was removed from the water bath and the reaction stopped by addition of an aliquot of chilled acetonitrile. Each sample was stored on ice, then treated using an ultrasonic bath to fully disrupt the cells.

INTERSPECIES COMPARISON INCUBATIONS
Based on the results of the preliminary incubations taking into account LDH leakage results, the incubations of [14C]test material in the interspecies comparison phase were conducted as follows:
- Hepatocyte species: Mouse, rat (two strains, namely Sprague-Dawley and Fischer 344), dog and human
- [14C]Test material nominal concentration: 10 µM
- Incubation times: 0.25 and 1 h
- Cell concentrations: Mouse: 0.3 x 10^6 viable cells/mL culture medium; Other species: 1 x 10^6 viable cells/mL culture medium.
- Incubation volume: 1 mL
- Number of replicates: One
- Incubation temperature: 37 °C

PROCESSING OF [14C]TEST MATERIAL HEPATOCYTE SAMPLES
Following sonication, duplicate aliquots of each incubate were taken for liquid scintillation counting (LSC). Each incubation sample was then centrifuged to sediment the cell debris. The resulting supernatant was transferred to an HPLC vial and duplicate aliquots were taken for LSC.

HPLC CONDITIONS FOR THE ANALYSIS OF PRELIMINARY INCUBATION SAMPLES
Instrumentation:
- System: Alliance 2695 Separations Module with built in pump, autosampler, membrane degassing system and column oven
- UV detector: 486 Tunable absorbance detector
- Radioactivity detector: β-RAM fitted with a 500 µL liquid cell, using Monoflow 4 scintillation cocktail (cocktail to mobile phase ratio of 3:1)
- Fraction collector: Gilson FC204
- Data collection/analysis: Laura software (version 1.4a or 4.1 (for fraction collection data processing))

Chromatographic conditions:
- Column: Capcell-Pak C18 UG120 (250 × 4.6 mm, 5 µm), fitted with a Phenomenex SecurityGuard C18 (4 × 3 mm)
- Mobile phase A: 0.1 % (v/v) Formic acid in water
- Mobile phase B: 0.1 % (v/v) Formic acid in acetonitrile
- Gradient: At 0 and 2 minutes: 95 % A, 5 % B; at 10 minutes: 60 % A, 40 % B; at 25 and 34 minutes: 0 % A, 100 % B; at 35 and 40 minutes: 95 % A, 5 % B.
- Oven temperature: 30 °C
- Flow rate: 1 mL/min
- UV detection: 230 nm

Preliminary incubation samples were analysed using the HPLC conditions stated above with on-line radiodetection with the exception of the 1, 3 and 10 µM samples for which off-line fraction collection (12 s fractions) into micro-plates followed by scintillation counting using a TopCount Microplate Scintillation Counter, was employed.

METABOLITE IDENTIFICATION
Individual solutions of each of the test material and O-10, O-34, OMT-B, O-70, O-72 and O-73 were prepared at 1 mg/mL. An aliquot (100 µL) of each was then combined to give a standard mix containing 0.14 mg/mL of each reference standard which was used for LC-MS analysis.
Instrumentation:
- HPLC pump: Agilent Quaternary pump G1311A
- Auto-sampler: Agilent ALS G1329A
- Degasser: Agilent Degasser G1322A
- UV detector: Agilent VWD G1314B
- Column heater: Agilent Column Oven G1316A
- Fraction collector: Gilson FC204
- Mass spectrometer: AB/MDS Sciex 4000 Qtrap
- Data system: Dell Optiplex (Windows XP SP3)
- Mass spectrometry software: Analyst v1.4.2 (AB/MDS)

HPLC conditions were as above but with a flow split ratio of ca 7:3. The majority went to the fraction collector (fractions collected at 0.1 min/well for 35 minutes) and the remainder to mass spectrometer after passing through the UV detector.

Mass Spectrometry conditions (enhanced mass scan):
- Ionisation: Electrospray ionisation (ESI), positive ion mode
- Spray voltage: 5 kV
- Temperature: 400 °C
- Curtain gas: Nitrogen at ca. 20 psi
- Ion source gas 1 and 2: Zero air at ca. 40 psi
- Scan range: 100 to 800 amu

MS/MS conditions (enhanced product ion):
- Collision energy: 40 eV
- Collision energy spread: 5 eV
- Collision gas: Nitrogen
- Scan range: m/z 50 to (M+10) amu (where M is the protonated molecular ion)

Liquid scintillation analysis:
Radioactivity measurements were performed in duplicate (where possible). Generally, aliquots of [14C]test material, incubation samples and HPLC eluent were mixed directly with Ultima Gold scintillation cocktail (5 mL) for liquid scintillation analysis. For metabolite profiling, radioactivity (cpm) was measured in HPLC fractions that had previously been collected into 96 DeepWell LumaPlates and dried in a centrifugal evaporator. A TopCount scintillation counter was used to count each plate, with multiple wells simultaneously counted for 5 minutes, after an initial 5 minute delay.

HPLC INTERPRETATION
For each chromatogram obtained, the regions of radioactivity were assigned metabolite fraction identities of M1 – M25 (each representing either a separated radioactive component or components where complete resolution could not be attained) and test material. Not all metabolite fractions were present in every sample and some were only present in trace quantities.
Following LC-MS analysis, in addition to test material, molecular weights were determined and structures postulated for seven of the 25 assigned radioactive regions. It was not possible to determine molecular mass for all metabolite fractions, namely those eluting with a retention time of earlier than 16 minutes. This may be due to poor ionisation of metabolites or their ready formation of adduct ions, or may be due to masking of the metabolites by endogenous substances. The characterisation of prominent metabolites by LC-MS/MS analysis was undertaken using samples generated from the incubation of [14C]test material with each of dog and human hepatocytes as these samples gave the strongest MS response.

Statistics:

None performed

Preliminary studies:

PRELIMINARY INCUBATIONS
The incubation of [14C]test material with Sprague-Dawley rat hepatocytes at nominal concentrations of 0.1, 0.5, 1, 5, 10, 30, 50 and 100 μM for 0, 0.25, 0.5, 1 and 3 h revealed that [14C]test material was possibly unstable under the experimental conditions used at low concentrations as indicated by the amount of radioactivity attributed to [14C]test material at 1 µM at the zero time point where the parent peak accounted for 58.3 % of the region of interest (ROI). The instability of [14C]test material is also indicated by the %ROI attributable to parent after 3 hours incubation in the absence of hepatocytes (parent ranged from 51.2 % at 1 µM to 93.9 % at 100 µM).
In the presence of rat hepatocytes, [14C]test material was extensively metabolised after 3 h incubation as exemplified by the depletion of parent (parent was reduced to below the limit of quantification (<1 % ROI)) at 3 and 10 µM [14C]test material (Table 1), whilst % depletion (compared to time zero) was 94.4 and 75.9 % at 30 and 100 µM [14C]test material, respectively, at the same time point.
Based on these data, main interspecies comparison incubations were conducted at a [14C]test material concentration of 10 µM for 0.25 and 1 h.

Metabolites identified:

yes

Details on metabolites:

RESULTS AND DISCUSSION
Between 0 h and 1 h, the highest leakage of LDH from cells incubated in the presence of [14C]test material was 20 % for 100 µM [14C]test material at 1 h (compared with 18 % for the solvent control at the same time point; note that 32 % LDH leakage was measured for the solvent control at 0.25 h which was probably a spurious result due to a sampling error at that time point). At 3 h, maximum leakage of 60 % was attained at 100 µM [14C]test material compared with 24 % for the solvent control. Overall, there was no notable difference between the LDH leakage from Sprague Dawley cells incubated in the absence of [14C]test material and in the presence of increasing concentrations of [14C]test material, with the exception of 100 µM [14C]test material after 3 h incubation. In the absence of hepatotoxicity (as indicated by LDH leakage) in Sprague-Dawley rat hepatocytes until 100 µM [14C]test material (3 h incubation), and the non-cytotoxicity in these hepatocytes at 30 µM [14C]test material for 3 hours, together with the absence of liver effects from in vivo studies with test material, where there was no indication that other species (mouse and dog) were more sensitive than the rat to the test material treatment, further investigation of the cytotoxicity of exposure to 10 µM [14C]test material for up to 1 hour for the other species was not considered necessary. The metabolite profiles produced for all species also indicated the absence of cytotoxicity, viz (i) the extents of metabolism observed for all species following incubation with 10 µM [14C]test material for 0.25 and 1 hour, and (ii) the number of metabolites formed during the course of these incubations.

INCUBATION OF [14C]TEST MATERIAL WITH MOUSE, RAT, DOG AND HUMAN CRYOPRESERVED HEPATOCYTES (INTERSPECIES COMPARISONS)
A comparison of metabolic profiles is presented in Table 2 below.
Following the incubation of [14C]test material (10 μM) for 0.25 and 1 h with hepatocytes from mouse, rat (from two strains: Sprague-Dawley and Fischer 344), dog and human, up to 25 radioactive regions were detected by radio-HPLC and assigned as metabolite fractions (M1 – M25) in addition to unchanged test material. Based on visual comparisons of the radio-HPLC traces it can be discerned that complete resolution of the radioactive components had not been attained in some samples, as in samples where the components were formed at lower levels, separation of the individual components was not achieved, suggesting that more than 25 metabolites of test material may have been formed in the present study.
With the exception of the two rat strains, which produced a greater proportion of radioactivity as metabolites with a retention time <15.1 minutes (particularly M2 and M7), the metabolite profiles obtained across the species were very similar (particularly at the 1 hour incubation time point). In all species M10 was either the predominant or one of the predominant metabolite fractions formed. Metabolite fractions M2 and M7 were major metabolite fractions in both strains of rat, but not in the other species investigated (mouse, dog and human). Although the radio-chromatograms showed quantifiable regions of radioactivity (≥1 % of the total chromatogram radioactivity), with some metabolite fractions comprising ≥20 % of the total chromatogram radioactivity, such as M2, it was not possible to determine molecular mass for all metabolite fractions; namely those eluting with a retention time of earlier than 16 minutes (metabolite fractions M2 - M8).
The most extensive metabolism of [14C]test material (based on the lowest percentage of total chromatogram radioactivity in the peak attributed to [14C]test material) was observed following 1 h incubation with rat (both Sprague-Dawley and Fischer 344; ca. 4 % [14C]test material remaining). There were no major differences observed in the metabolite profiles between the two strains of rat under the experimental conditions used, although the Fischer 344 rat produced less pronounced peaks for M8, M11, M12 and M19 compared to the Sprague-Dawley rat.
Conversely, following 1 h incubation, the least metabolism (based on the highest percentage of total chromatogram radioactivity in the peak attributed to [14C]test material) was observed in mouse (33.0 % [14C]test material remaining) and human (34.4 % [14C]test material remaining). In the mouse, metabolite fractions with a retention time of <16 minutes (M2 – M8) were only present at trace amounts or absent. Similarly for human hepatocytes, of metabolites with a retention time of <16 minutes only M2 and M6 were present in trace amounts. In comparison, the dog metabolised [14C]test material quite extensively (with ca. 16 % [14C]test material remaining), although compared to the two rat strains, M2 and M7 were absent and M8 was present at only a trace amount. Compared to other the species M16 was most prominent in the dog.
Following LC-MS analysis, in addition to test material, molecular weights were determined and structures postulated for seven of the 25 assigned radioactive regions. It was not possible to determine molecular mass for all metabolite fractions, namely those eluting with a retention time of earlier than 16 minutes. This may be due to poor ionisation of metabolites or their ready formation of adduct ions, or may be due to masking of the metabolites by endogenous substances. From the LC-MS(MS) data, it can be concluded that the test material undergoes both Phase I and Phase II metabolism via hydroxylation, hydrolysis, oxidation and glucuronide conjugation reactions.
The following assignations were considered possible:
M9: a dihydroxylated metabolite of the test material.
M12: a glucuronide conjugate of hydroxylated test material.
M15: a carboxylated metabolite of the test material.
M16, M17 and M18: hydroxylated metabolites of the test material.
M19: Test material with prop-2-yn-1-yl sulfite cleaved away (reference standard O-10).
However, the two major in vitro metabolites of the test material (metabolites M2 and M10) remain unknown.

Table 1: Percentage depletion of [14C]Test Material following Incubation with Sprague-Dawley Rat Hepatocytes

Concentration (μM)	% [14C]Test Material depletion				%ROI with hepatocytes	%ROI in no hepatocyte samples
	Time (hours)
	0.25	0.5	1	3	0	3
1	54.4	56.3	57.1	57.2	58.3	51.2
3	94.6	92.3	BLQ	BLQ	94.9	30.1
10	68.4	88.4	97.3	BLQ	99.0	80.2
30	34.9	72.8	86.2	94.4	95.4	90.6
100	27.2	41.0	71.4	75.9	98.0	93.9

ROI = Region of interest

BLQ = Below the limit of quantification - % ROI accounting for less than 1 % of the total radioachromatogram attributable to [14C]test material

Table 2: Metabolite fractions detectedfollowing 0.25 and 1 h incubations of[14C]Test Material(10 µM) with mouse, rat, dog and human hepatocytes

Metabolite fraction	RT (min)	Percentage of total chromatogram radioactivity in metabolite fraction
		Mouse		Sprague-Dawley rat		Fischer 344 rat		Dog		Human
		0.25 h	1 h	0.25 h	1 h	0.25 h	1 h	0.25 h	1 h	0.25 h	1 h
M1	3.9	-	-	-	-	-	-	1.7	-	-	-
M2	12.4	-	1.3	2.8	20.3	3.4	30.6	-	-	-	1.1
M3	12.9	2.5	-	-	-	-	-	-	-	-	-
M4	13.5	1.9	1.9	-	-	-	-	-	-	-	-
M5	13.9	-	1.7	-	3.8	2.4	5.7	-	5.2	-	-
M6	14.2	-	1.7	1.4	3.1	1.2	-	-	6.3	-	2.3
M7	15.1	-	-	-	5.6	2.1	8.9	-		-	-
M8	15.7	-	2.7	1.5	10.0	-	3.0	-	1.1	-	-
M9	16.7	1.4	5.9	3.3	5.4	3.4	6.0	1.1	8.0	2.0	5.8
M10	17.6	2.2	10.3	12.5	20.0	11.3	18.3	2.7	20.6	3.4	15.7
M11	18.4	1.1	4.5	-	1.9	2.3	-	-	-	-	-
M12	18.7	-	1.5	2.6	2.8	-	-	-	2.3	1.3	9.7
M13	19.0	-	-	-	-	1.3	-	-	2.5	-	-
M14	20.0	-	-	-	1.6	-	1.8	-	-	-	-
M15	20.6	1.3	2.5	-	1.3	-	1.4	-	3.9	-	-
M16	21.1	1.5	2.6	1.2	-	1.1	-	4.3	7.7	-	1.3
M17	21.8	2.0	2.7	1.3	1.0	1.4	-	2.0	4.6	3.3	6.1
M18	22.2	3.0	3.6	3.4	-	3.8	-	2.1	4.6	10.3	8.1
M19	23.0	2.8	6.4	7.6	2.4	7.7	1.0	3.3	4.1	3.2	4.3
M20	23.2	-	-	-	-	1.8	-	-	-	-	-
M21	23.5	-	-	-	-	2.4	-	-	-	-	-
M22	24.0	1.0	-	-	-	-	-	-	-	-	-
Test Material	25.9	69.8	33.0	47.1	4.0	28.0	3.9	73.9	16.2	57.4	34.4
M23	26.4	-	3.0	1.8	1.7	12.5	1.1	-	-	5.7	-
M24	26.7	-	-	-	-	3.9	1.1	-	-	-	-
M25	27.3	-	-	-	-	-	-	-	-	2.3	-
Rem	NA	9.5	14.7	13.4	15.1	10.0	17.2	8.9	12.9	11.1	11.2

- = Not detected, accounting for <1 % of total chromatogram radioactivity

NA = Not applicable

Rem = Remainder of radioactivity associated with diffuse areas in the chromatogram

RT = Typical retention time

Conclusions:

Interpretation of results: bioaccumulation potential cannot be judged based on study results
Overall, the test material was very rapidly and extensively metabolised, especially by the two strains of rat used in this study. In all species M10 was either the predominant or one of the predominant metabolite fractions formed. The most extensive metabolism of [14C]test material (based on the lowest percentage of total chromatogram radioactivity in the peak attributed to [14C]test material) was observed following 1 h incubation with rat (both Sprague-Dawley and Fischer 344). Equally there were no major differences observed in the metabolite profiles between these two different strains under the experimental conditions used. Conversely, following 1 h incubation, the least metabolism (based on the highest percentage of total chromatogram radioactivity in the peak attributed to [14C]test material) was observed in mouse and human. Metabolite fraction M2 and M7 were major metabolite fractions in both strains of rat, but not in the other species investigated (mouse, dog and human).
From the available LC-MS(MS) data, it can be concluded that the test material undergoes both Phase I and Phase II metabolism via hydroxylation, hydrolysis, oxidation and glucuronide conjugation reactions. However, the two major in vitro metabolites of the test material (metabolites M2 and M10) remain unknown.

Executive summary:

The purpose of this study was to compare the metabolism in vitro of [14C]test material using hepatocytes from mouse, rat (two strains, namely Sprague-Dawley and Fischer 344), dog and human under GLP conditions.

After initially confirming the solubility of the test material in acetonitrile (10 mM) and then following 100-fold dilution in incubation media (final concentration: 100 μM) by visual inspection, preliminary incubations of [14C]test material (nominal concentrations: 1,3,10, 30 and 100 µM) were conducted with pooled male and female Sprague-Dawley rat cryopreserved hepatocytes (1 x 10⁶ viable cells) for 0, 0.25, 0.5, 1 and 3 hours, each as a single sample in order to check the metabolic stability of [14C]test material. At the end of the incubation period, reactions were terminated by the addition of chilled acetonitrile and disruption of the cells in the incubation medium by sonication. Samples were subsequently centrifuged in order to pellet any cell debris present. Resulting supernatants were analysed by reversed-phase HPLC with either on-line radiodetection (30 and 100 μM samples) or off-line fraction collection and scintillation counting via TopCount (1, 3 and 10 μM samples). The potential cytotoxicity of [14C]test material was assessed by the measurement of the leakage of lactate dehydrogenase (LDH) following incubation of [14C]test material with pooled Sprague-Dawley rat hepatocytes under the incubation conditions described above, with the inclusion of a solvent control sample.

Interspecies comparison incubations of [14C]test material metabolism (nominal concentration: 10 μM) using cryopreserved hepatocytes from pooled male and female mouse (0.3 x 10⁶ viable cells/mL), rat (separately from each of Sprague-Dawley and Fischer 344), dog and human (1 x 10⁶ viable cells/mL) was subsequently conducted at incubation times of 0.25 and 1 hour, in a similar manner to the preliminary incubation. Samples were processed as for the preliminary incubation. Sample supernatants were analysed by LC-MS with off-line fraction collection and scintillation counting via TopCount.

In the preliminary incubations [14C]test material was extensively metabolised by Sprague-Dawley rat cryopreserved hepatocytes after 3 hours incubation as exemplified by the depletion of parent (%ROI attributable to parent was reduced to below the limit of quantification (<1 % ROI)) at 3 and 10 μM [14C]test material after 3 hours incubation, whilst % depletion (compared to time zero) was 94.4 and 75.9 % at 30 and 100 μM [14C]test material, respectively, at the same time point. Based on these results, a [14C]test material concentration of 10 μM and timepoints of 0.25 and 1 hour were selected for the interspecies comparison incubations. The measurement of lactate dehydrogenase (LDH) leakage from Sprague-Dawley rat hepatocytes incubated with [14C]test material at 0, 1, 3, 10, 30 and 100 μM for either 0, 0.25, 0.5, 1 and 3 hours indicated that there was only a marked increase in the LDH leakage following the incubation of [14C]test material at 100 μM for 3 hours compared to the solvent control at the same time point (from 24 % at 0 μM [14C]test material to 60 % at 100 μM [14C]test material).

In the main interspecies comparison incubations, the incubation of [14C]test material at a final concentration of 10 µM with pooled male and female mouse, rat (each of Sprague-Dawley and Fischer 344), dog and human cryopreserved hepatocytes for 0.25 and 1 hours revealed that [14C]test material was metabolised to a maximum of 25 potential metabolite fractions (designated M1 to M25). 

The metabolite profiles obtained across the species were very similar, with M10 being either the predominant or one of the predominant metabolite fractions formed. Metabolite fraction M2 was a major metabolite fraction in both strains of rat, but not in human or the other toxicological species investigated (mouse and dog). Although the radio-chromatograms showed quantifiable regions of radioactivity (≥1 % of the total chromatogram radioactivity), with some metabolite fractions comprising ≥20 % of the total chromatogram radioactivity, such as M2, it was not possible to determine molecular mass for all metabolite fractions; namely those eluting with a retention time of earlier than 16 minutes.

Overall, the test material was very rapidly and extensively metabolised, especially by the two strains of rat used in this study. In all species M10 was either the predominant or one of the predominant metabolite fractions formed. The most extensive metabolism of [14C]test material (based on the lowest percentage of total chromatogram radioactivity in the peak attributed to [14C]test material) was observed following 1 h incubation with rat (both Sprague-Dawley and Fischer 344). Equally there were no major differences observed in the metabolite profiles between these two different strains under the experimental conditions used. Conversely, following 1 h incubation, the least metabolism (based on the highest percentage of total chromatogram radioactivity in the peak attributed to [14C]test material) was observed in mouse and human. Metabolite fraction M2 and M7 were major metabolite fractions in both strains of rat, but not in the other species investigated (mouse, dog and human).

From the available LC-MS(MS) data, it can be concluded that the test material undergoes both Phase I and Phase II metabolism via hydroxylation, hydrolysis, oxidation and glucuronide conjugation reactions. However, the two major in vitro metabolites of the test material (metabolites M2 and M10) remain unknown.

Description of key information

Full data package investigating the adsorption, distribution, metabolism and excretion of propargite.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

66

Additional information

Propargite is rapidly and extensively absorbed, metabolised and eliminated from the bodies of rats and mice. At low dose levels, peak urinary excretion is typically within 0-24 hours of dosing and accounts for around 50-60 % of administered dose, whereas peak faecal excretion typically occurs within 0-48 hours and accounts for around 35-60 % of administered dose. Urinary and faecal elimination is usually near complete (>93 %) by 48 and 72 hours post-dose respectively. The actual extent/rate/route of absorption and elimination is dependent upon sex, dose level and species.

At relatively low dose levels (25-150 mg/kg bw), urinary excretion appears to be greater in male rats than in females (59.2-61.1 % versus 43.6-49.8 %) whereas faecal excretion appears to be greater in females than males (50.9-61.2 % versus 35.4-51.3 %). This sex-dependent pattern is also apparent in mice, with urinary excretion accounting for 58.8 and 47.1 % of the administered dose in males and females, respectively, and faecal excretion accounting for 41.5 % (males) and 52.9 % (females). At higher doses, this sex-related difference is less apparent (e.g. 30.3 % males, 34.4 % females for urinary excretion and 74.5 % males and 69.9 % females for faecal excretion at 200 mg/kg bw). This illustrates the fact that, at higher dose levels, urinary excretion becomes less important than faecal excretion and the time to peak urinary excretion becomes longer, perhaps occurring at 36-96 hours. A further study showed urinary excretion accounting for 34.5, 32.9 and 21.2 % of administered dose in rats receiving single oral doses of 25, 60 and 200 mg/kg bw, respectively, whereas corresponding faecal values were 48, 70.9 and 87.7 %. These findings suggest that absorption and urinary excretion pathways can become saturated at high doses, with excretion of unabsorbed propargite in the faeces becoming a more important route of elimination for orally administered material. In a study that specifically compared rats and mice, urinary excretion was a more important route of elimination in mice than rats, accounting for 39.9 and 24.9 % of administered dose, respectively. Corresponding values for faecal elimination were 29.2 and 48.1 %.

Accumulation in internal organs appears to be negligible, with the majority of residues remaining in the G.I. tract/contents and/or carcass. For example, values for accumulation as percentage of administered dose of 9.39 % (72 hours, single dose of 270 mg/kg bw), 1.5, 1.6 and 23.3 % (96 hours, single doses of 25, 60 and 200 mg/kg bw, respectively) and 0.6 % (repeat oral dose, 25 mg/kg bw, males) have been reported for rats. However, of these values, 3.78/5.05, 0.7, 0.82, 2.39 and 0.1/0.29 % of administered dose was present in G.I. tract/carcass, G.I. tract and contents, G.I. tract and contents, G.I. tract and contents and G.I. tract and contents/carcass, respectively. This pattern of distribution is further illustrated in a study in which rats were administered the high oral dose of 1250 mg/kg bw. In this study, of the retained 53.3 % radioactivity, 49.38 % was present in the G.I. tract and contents, 3.4 % in the carcass with <1 % actually administered in the internal organs. From these findings, it can be concluded that typically less than 1 % of administered propargite is accumulated in the internal tissues and organs of mammals 72 to 96 hours after oral exposure.

The majority of radioactivity associated with the G.I. tract and faeces is as unabsorbed propargite. However, biliary excretion of metabolites arising from absorbed propargite may also contribute to the radioactivity observed in the intestines, as indicated in a study in which bile collected from rats and mice contained respectively 15.5 and 14.8 % of an administered dose of 150 mg/kg bw. This would indicate that of a typical 30-60 % faecal excretion of radioactivity, 20-45 % could be present as unchanged propargite with the remainder being metabolites coming from biliary excretion.

On the basis that oral absorption is equal to total urinary excretion and biliary excretion and tissue accumulation, then oral absorption of propargite could theoretically be as high as 76 % in male rats (60 + 15 + 1 %). Since urinary excretion is typically 10 % lower in female rats (and faecal excretion is correspondingly approximately 10 % greater), it can be concluded that oral absorption in female rats is lower, at up to 66 %.

Analysis indicates that the relative proportions of metabolites in excreta is dose, sex and species dependent (for example, propargite is more extensively metabolised by mice than rats). However, it can be concluded that propargite is metabolised by hydrolysis of the propynyl sulphite side chain and subsequent oxidation of a methyl group of the tert-butyl portion and hydroxylation of the cyclohexyl moiety to yield HOMe-TBPC-diol and HOMe-TBPC-triol. Hydroxylation of the cyclohexyl moiety produces a mixture of positional and/or stereochemical (axial/equatorial) hydroxy isomers. These metabolites in turn undergo conjugation with sulphate to produce HOMe-TBPC-diol sulphate and HOMe-TBPC-triol sulphate. Further oxidation of the hydroxylated tert-butyl groups produced Carboxy-TBPC type metabolites.

A new in vitro comparative ADME study was completed in 2015 (Cheung, 2015). The most extensive metabolism of [14C]Propargite (based on the lowest percentage of total chromatogram radioactivity in the peak attributed to [14C]Propargite) was observed following 1 h incubation with rat hepatocytes (both Sprague-Dawley and Fischer 344; ca. 4 % [14C]Propargite remaining). The rat appeared to metabolise propargite faster than the mouse, dog or human. There were no major differences observed in the metabolite profiles between the two strains of rat. With the exception of the two rat strains, which produced a greater proportion of radioactivity as metabolites with a retention time <15.1 minutes (particularly M2 and M7), the metabolite profiles obtained across the species were qualitatively very similar (particularly at the 1 hour incubation time point). In all species M10 was either the predominant or one of the predominant metabolite fractions formed. Metabolite fractions M2 and M7 were major metabolite fractions in both strains of rat, but not in the other species investigated (mouse, dog and human).