ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

3 - 7 Apr 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

preliminary study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

Deviations to the current guideline (adopted in 2010): low number of animals used, i.e. 2 males and 2 females at the low dose, and 1 male and 1 female at the high dose; no detailed identification of metabolites.

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C labelled test substance

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, UK
- Age at study initiation: 7 weeks
- Body weight at study initiation: elimination/tissue residue examinations: males: 152 - 158 g, females: 140 - 147 g; blood level examinations: males: 202 - 283 g, females: 158 - 283 g
- Fasting period before study: yes, for 16 h before dosing
- Housing: During acclimatisation and up to dosing, rats were housed in single sex groups, in plastic holding cages with wood chip bedding.
- Individual metabolism cages: yes, after dosing rats subjected to elimination/tissue residue study were tranferred to individual all glass metabolism cages; rats used for blood level studies were housed in individual plastic cages after dosing.
- Diet: pelleted laboratory rodent diet CRM (P) SQC (Special Diet Services, Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 Apr 1995 To: 07 Apr 1995

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
10 mg/kg bw: [14C]-HOE 122 006 prepared at a nominal concentration of 2 mg/mL and specific activity 20 µCi/mg (dissolving the labelled and unlabelled HOE 122 006 in sesame oil)
500 mg/kg bw: [14C]-HOE 122 006 prepared at a nominal concentration of 100 mg/mL and specific activity 0.5 µCi/mg (co-precipitation of radiolabelled and unlabelled HOE 122 006 from chloroform followed by suspension in sesame oil)

VEHICLE
- Justification for use and choice of vehicle: The test substance was dissolved/suspended in the same vehicle employed for other preliminary toxicology studies performed with the test substance.
- Concentration in vehicle: 2 mg/mL (low dose 10 mg/kg bw), 100 mg/mL (high dose, 500 mg/kg bw)
- Amount of vehicle: 5 mL/kg bw

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
No impurities were noted, except in the low dose, post-dosing sample, where a small amount (3.48%) of degradation product was noted. This was considered an acceptable level post-dosing, therefore the dose solutions were deemed stable during the dose formulation and study procedures.

Duration and frequency of treatment / exposure:

single treatment

Dose / conc.:

10 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

10 mg/kg bw: 4 males, 4 females; two animals of each sex were used for elimination/tissue residue examinations, the other two of each sex were used for blood level examinations
500 mg/kg bw: 2 male, 2 female; one animal of each sex was used for elimination examinations, the remaining animal of each sex was used for blood level examinations

Cmax study: 200 mg/kg bw: 1 female

Control animals:

no

Details on study design:

- Dose selection rationale: Animals were dosed at 10 mg/kg bw, a no observable effect level in the rat, and 500 mg/kg bw, a level that produced toxic effects in subchronic studies.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, adrenals, bone (right hind leg), brain, eyes, gastro-intestinal tract, heart, kidneys, liver, lungs, muscle (right hind leg), ovaries, renal fat, spleen, testes, thyroid and residual carcass. In addition, cage washes were collected.
Gastro-intestinal tract and carcass were processed immediately after necropsy. All other samples (excreta, tissues and organs) were stored deep frozen until analysis.
- Time and frequency of sampling:
Urine and faeces: 24, 48, 72, and 96 h post dosing. After sample collection the metabolism cages were rinsed with water. These cage washes were kept either separately or together with urine samples.
Necropsy: 96 h post dosing
Plasma: 96 h post dosing
Blood: 0.25, 0.5, 1, 1.5, 2, 4, 6, 24, 30, 48, 72, and 96 h post dosing (collected by tail snipping and bleeding onto weighed absorbant pads)
Parent compound at tmax: aortic exsanguination. Blood samples were retained for analysis.
Expired air: [14C]-CO2 traps were assayed after 24 h and shown to contain less than 2% of the administered radioactivity. The trapping was therefore discontinued after this time.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled : urine, faeces
- Time and frequency of sampling: 24 h
- From how many animals: 10 mg/kg bw: 2 males, 2 females, 500 mg/kg bw: 1 male, 1 female; samples not pooled
- Method type(s) for identification: HPLC-UV, TLC, liquid scintillation counting
- Limits of detection and quantification: depending on tissue type, for 10 mg/kg bw: 0.005 - 0.054 mg/kg, for 500 mg/kg bw: 0.164 - 1.070 mg/kg, whereas the limit of detection corresponded to dpm in sample equivalent to background cpm level

TREATMENT FOR CLEAVAGE OF CONJUGATES:
SHP (Helix pomatia juice, Sepracos, USA; 0.25 mL) was added to urine (0.5 mL) in acetate buffer (0.2 M; pH 5; 20 mL). The mixture was incubated for 18 h at 37 °C.

Statistics:

Elimination/tissue residue examinations
Percentage dose present and concentration in all samples were calculated. Means and standard deviations for each analysis and for individual values in each group were also calculated.
Blood examinations
Concentration of total radioactivity in blood samples was calculated. Means and standard deviations for each analysis was calculated where
appropriate.

Pharmacokinetic parameters were determined using the Topfit 2.0 pharmacokinetic program. Blood concentration versus time profiles were fitted using a two compartment disposition model.

Type:

absorption

Results:

The test substance was quickly absorbed i.e. the maximum concentration of radioactivity in blood was found 1 h (females) to 4 h (males) after administration of 10 mg/kg bw and at 4 h after administration of 500 mg/kg bw in females and males.

Type:

distribution

Results:

At all time-points examined, radioactivity (of radiolabelled test substance) in most tissue was beneath the limit of quantification. Radioactivity was mainly observed in the excretory organs kidney and liver and in the carcass/fatty tissues.

Type:

metabolism

Results:

The test substance was extensively metabolised, and only trace amounts of parent compound were detectable in the faeces, with none in either urine or plasma. The acid derivative of the parent ester was the major metabolite.

Type:

excretion

Results:

The test substance was eliminated from the body predominantly via urine (70%). Excretion (urine and faeces) was almost complete (85-90%) 24 h and 48 h post dosing for low and high dose animals, respectively. Expiration of radiolabelled CO2 was <2%.

Details on absorption:

Absorption following dosing at 10 mg/kg bw was quicker in females (1.0 h to reach maximum blood concentration) compared to males (4.0 h). After dosing with 500 mg/kg bw, absorption was slowed such that maximum blood concentration was not reached until 4.1 h (male and female). Blood concentrations of radiolabelled test substance declined biphasically, with terminal half-lifes of 38.9 h (male) and 32.3 h (female) from animals dosed at 10 mg/kg bw and 14.4 h (male) and 30.1 h (female) from animals dosed at 500 mg/kg bw. The female low dose blood concentration/time curves showed a far higher clearance than the male low dose animals (approximately 8-fold difference) and a much reduced AUC (approximately 7-fold lower). In the high dose animals, these sex differences were not seen, as the clearance and AUC figures were similar for both sexes.

Details on distribution in tissues:

Tissue residues of the radiolabelled test substance 96 h post dosing were mainly below the level of quantification in the low dose animals. The organs with detectable levels of residues were the liver (0.112 and 0.037 µg/g tissue for males and females, respectively) and kidney (0.359 and 0.131 µg/g tissue for males and females, respectively). Males had a three-fold greater tissue concentration of residues than females. In the high dose animals, residues were found in a greater number of tissues, but still at very low levels. Again the liver (2.281 and 3.806 µg/g tissue for males and females, respectively) and kidney (1.997 and 2.364 µg/g tissue for males and females, respectively) showed the highest concentrations of residues, but residues were also detected in renal fat (1.050 and 2.086 µg/g tissue, males and females, respectively).

Details on excretion:

Urinary excretion accounted for 70% of the eliminated radiolabel, except in the low dose males where it accounted for 50% of elimination. The rest of the radiolabel was recovered in the faeces. Elimination of radiolabelled test substance was rapid, with 90% of the dose excreted in either faeces or urine after 24 hours in the low dose animals. In the high dose animals excretion after 24 hours accounted for 80% of the radiolabelled dose administered. Here, elimination of >90% was prolonged to 48 h.

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 10 mg/kg bw: male: 14.05 mg/kg bw, female: 3.84 mg/kg bw; 500 mg/kg bw: male: 279 mg/kg bw, female: 236 mg/kg bw

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 10 mg/kg bw: male: 4.0 h, female: 1.0 h; 500 mg/kg bw: male: 4.1 h, female: 4.1 h

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 10 mg/kg bw: male: 38.9 h, female: 32.3 h; 500 mg/kg bw: male: 14.4 h, female: 30.1 h

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 10 mg/kg bw: male: 168.51 mg × h/kg, female: 22.75 mg × h/kg; 500 mg/kg bw: male: 4176 mg × h/kg, female: 4233 mg × h/kg

Metabolites identified:

yes

Details on metabolites:

The test substance was extensively metabolised at both dose levels, and only trace amounts of parent compound were detectable in the faeces, with none in either urine or plasma. The major metabolite at the high dose was putatively identified as the acid derivative of the parent ester. At the low dose there was a marked quantitative sex difference in the metabolism profile. In females, the profile was similar to that seen at the high dose (mainly the acid metabolite) but in males more polar metabolite(s) were present which accounted for >50% of the radioactivity. This may account for the faster initial half-life in the blood, and the greater clearance seen in male animals.

Conclusions:

The test substance was readily absorbed following oral administration and rapidly distributed mainly to liver and kidney. Analysis of metabolites revealed extensive metabolism of the test substance. Excretion, mainly via urine and to a lesser extend via faeces, was completed 48 h post dosing. No bioaccumulation potential was observed after single administration.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 - 15 Jun 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

Only one low dose was tested, one high dose was tested in a separate study (M-142589-01-1). Metabolites were characterised in a separate study (M-184537-01-1).

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C labelled test substance

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, UK
- Age at study initiation: 6 - 7 weeks
- Body weight at study initiation: males: 160 - 180 g, females: 156 - 165 g
- Fasting period before study: yes, for 16 h before dosing
- Housing: During acclimatisation and prior to dosing with the radiolabelled test substance, the rats were housed in single sex groups, in plastic holding cages with wood chip bedding.
- Individual metabolism cages: yes, all-glass metabolism cages, all animals post dosing
- Diet: pelleted laboratory rodent diet SDS CRM (P) (Special Diet Services, Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Jun 1995 To: 15 Jun 1995

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
10 mg/kg bw: [14C]-radiolabelled test substance was prepared at a nominal concentration of 2 mg/mL and specific activity 20 µCi/mg, dissolving the labelled and unlabelled test substance in sesame oil

VEHICLE
- Justification for use and choice of vehicle: Sesame oil was used as preliminary toxicology studies were conducted using it as the dosing vehicle for gavage studies.
- Concentration in vehicle: 2 mg/mL
- Amount of vehicle: 5 mL/kg bw

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The purity of the test substance in the dose solution was greater than 97% both pre- and post-dosing. The dose solution was deemed stable during the formulation and dosing procedures.

Duration and frequency of treatment / exposure:

single treatment

Dose / conc.:

10 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

5

Control animals:

no

Details on study design:

- Dose selection rationale: In previous toxicological studies 10 mg/kg bw were determined as the no observable effect level (NOEL).

Details on dosing and sampling:

PHARMACOKINETIC STUDY (absorption, distribution, excretion)
- Tissues and body fluids sampled:
urine, faeces, blood, plasma, adrenals, bone (right hind leg), brain, eyes, gastro-intestinal tract, heart, kidneys, liver, lungs, muscle (right hind leg), ovaries, renal fat, spleen, stomach, testes, thyroid, and residual carcass. In addition, cage washes were collected.
Gastro-intestinal tract and carcass were processed immediately after necropsy. All other samples (excreta, tissues and organs) were stored deep frozen until analysis.
- Time and frequency of sampling:
Urine: 6, 12, 24, and 48 h post dosing
Faeces: 24 and 48 h post dosing. After sample collection the metabolism cages were rinsed with water. These cage washes were kept separately.
Necropsy: 48 h post dosing
Blood (and plasma): taken at necropsy, 48 h post dosing

DETECTION METHOD:
Radioactivity was measured by liquid scintillation counting with automatic external standard quench correction. Corrected dpm of less than the background cpm were considered to be below the limit of quantification.

Statistics:

The concentration of tissue residues in all samples was calculated. Means and standard deviations for each analysis and for individual values in each group were calculated.

Type:

absorption

Results:

The minimum absorption of the test substance was estimated by the total urinary excretion. The total urinary excretion was 45.9% in males and 81.4% in females, 48 h after oral administration of 10 mg/kg bw radiolabelled test substance.

Type:

distribution

Results:

Sex differences were noted in the tissue residues at necropsy with males showing generally higher residue levels than females. Radioactivity was mainly observed in the excretory organs kidney, liver and in females only in renal fat.

Type:

excretion

Results:

The test substance was rapidly eliminated from the body, with an observed sex difference. 48 h post dosing excretion via faeces (47% and 11%) and urine (46% and 81%) was almost complete in males and females, respectively.

Details on absorption:

The total amount of a dose that is excreted via the urine is a surrogate for the minimum amount that has been absorbed. Following the administration of a single dose of 10 mg/kg body weight [14C]-radiolabelled test substance, male and female urinary excretion after 48 h was 41.69 ± 10.34% and 76.36 ± 10.52% of administered dose, respectively. A further 4.19 ± 2.23% and 5.04 ± 2.50% was recovered in cage washes for the male and female rats, respectively. Therefore, the minimum total absorption for male and female rats was 45.88 ± 10.94% and 81.40 ± 9.53%, respectively.

Details on distribution in tissues:

Differences in distribution were noted between the sexes, with males generally showing higher residue levels than females. Highest residue levels in males were seen in the kidney and liver (0.712 ± 0.343 and 0.311 ± 0.149 mg equivalent test substance/kg tissue, respectively). As these tissues are involved in the metabolism and elimination of the test substance, they are expected to show high residue levels. Residue levels in the heart and skin of male rats were 0.225 ± 0.064 and 0.169 ± 0.059 mg/kg tissue, respectively. With the exception of the stomach and gastro-intestinal tract, all other residue levels were below 0.1 mg/kg tissue.
In females, residue levels were highest in kidney, renal fat and liver (0.568 ± 0.502, 0.195 ± 0.047, and 0.163 ± 0.016 mg/kg tissue, respectively). The measured residue levels in renal fat suggest a degree of distribution into fatty tissues, which is more extensive in females than males. Residue levels in the muscle and gastro-intestinal tract of females were 0.133 ± 0.066 and 0.113 ± 0.058 mg/kg tissue, respectively. The gastro-intestinal tract residue levels are ten-fold lower in females compared to males, reflecting the greater faecal elimination by males compared to females.
No residues were detectable in the brain, thyroid and spleen of both sexes, and the plasma and eyes of the female rats. Lowest detectable residue levels in both sexes were in the adrenals and bone (0.010 ± 0.015 and 0.024 ± 0.004 mg/kg tissue, respectively for males, and 0.013 ± 0.018 and 0.002 ± 0.003 mg/kg tissue, respectively for females).

Details on excretion:

In males, excretion is via both the urine and faeces (41.69 ± 10.34 and 47.39 ± 10.95% of administered dose, respectively). The majority of the urinary excretion occurs within the first 12 h (68% of the total excreted by this route), and the majority of the faecal excretion occurs within the first 24 h (80% of the total excreted by this route). Total radioactivity recovered in the excreta by 48 h post dosing in males was 93.27 ± 2.347%.
In females, the excretion profile is different. The major route of excretion is via the urine (76.36 ± 10.52% of the administered dose after 48 h), with the faeces accounting for only 11.48 ± 8.35% of the administered dose. The majority of the dose excreted in the urine is recovered within 6 h post dosing (60% of the total excreted by this route), while 89% of the total eliminated in the faeces is recovered within the first 24 h. Total radioactivity recovered in the excreta by 48 h post dosing in females was 92.88 ± 1.99%.

Metabolites identified:

not measured

Table 1: Tissue distribution of radiolabelled residues following single oral dosing at 10 mg/kg bw

	Concentration (mg equivalent test substance/kg tissue)
	males		females
Tissue	Mean	SD	Mean	SD
Adrenals	0.010	0.015	0.013	0.018
Blood	0.053	0.013	0.016	0.002
Bone	0.024	0.004	0.002	0.003
Brain	BLQ	-	BLQ	-
Carcass	0.118	0.022	0.079	0.022
Eyes	0.006	0.004	BLQ	-
G.I. tract	1.230	0.511	0.113	0.058
Heart	0.225	0.064	0.035	0.012
Kidney	0.712	0.343	0.568	0.502
Liver	0.311	0.149	0.163	0.016
Lungs	0.028	0.022	0.023	0.022
Muscle	0.038	0.023	0.133	0.066
Ovaries	-	-	0.024	0.008
Plasma	0.051	0.017	BLQ	-
Renal fat	0.059	0.015	0.195	0.047
Skin	0.169	0.059	0.031	0.013
Spleen	BLQ	-	BLQ	-
Stomach	0.142	0.214	0.054	0.046
Testes	0.090	0.021	-	-
Thyroid	BLQ	-	BLQ	-

BLQ = Below level of quantification (mean dpm for sample is < twice background dpm).

Table 2: Elimination of radiolabelled material following single oral dosing at 10 mg/kg bw

		Percentage of radioactive dose
		Males		Females
		Mean	SD	Mean	SD
Urine	6h	16.25	8.75	45.45	9.39
	12h	11.95	3.45	17.33	2.24
	24h	9.59	1.78	11.82	2.78
	48h	3.91	1.07	1.76	0.87
	Sub-total	41.69	10.34	76.36	10.52
Faeces	24h	37.79	8.357	10.22	7.84
	48h	9.596	3.17	1.26	0.84
	Sub-total	47.39	10.95	11.48	8.35
Cage wash	24h	3.39	2.19	4.17	2.08
	48h	0.791	0.235	0.87	0.433
	Sub-total	4.19	2.23	5.04	2.5
% Total eliminated		93.27	2.347	92.88	1.987
% Carcass		0.958	0.167	0.629	0.176
% Total recovery		94.23	2.36	93.51	1.97

Conclusions:

Based on the results of the present study, the test substance was rapidly absorbed and eliminated by rats. There was a sex difference in the magnitude of the tissue residues and in the route of excretion. No bioaccumulation potential was observed after single administration.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 - 11 Jul 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

absorption

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

Only one high dose was tested, one low dose was tested in a separate study (M-142588-01-1). Metabolites were characterised in a separate study (M-184537-01-1).

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C labelled test substance

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, UK
- Age at study initiation: 6 - 7 weeks
- Body weight at study initiation: males: 170 - 181 g, females: 160 - 174 g
- Fasting period before study: yes, for 16 h before dosing
- Housing: During acclimatisation and prior to dosing with the radiolabelled test material, the rats were housed in single sex groups, in plastic holding cages with wood chip bedding.
- Individual metabolism cages: yes, all-glass metabolism cages, all animals post dosing
- Diet: pelleted laboratory rodent diet SDS CRM (P) (Special Diet Services, Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
500 mg/kg bw: [14C]-radiolabelled test substance was prepared at a nominal concentration of 100 mg/mL and specific activity of 0.5 µCi/mg, radiolabelled and unlabelled test substance was dissolved in chloroform, blown to dryness under a stream of dinitrogen gas, and desicated under vacuum overnight before dissolving in sesame oil

VEHICLE
- Justification for use and choice of vehicle: Sesame oil was used as preliminary toxicology studies were conducted using it as the dosing vehicle for gavage studies
- Concentration in vehicle: 100 mg/mL
- Amount of vehicle: 5 mL/kg bw

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The purity of the test substance in the dose solution was greater than 97% both pre- and post-dosing. Thus the dose solution was deemed stable during the formulation and dosing procedures.

Duration and frequency of treatment / exposure:

single treatment

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

actual dose received: 456.8 ± 12.7 mg/kg bw

No. of animals per sex per dose / concentration:

5

Control animals:

no

Details on study design:

- Dose selection rationale: 500 mg/kg bw was identified as the highest dose level that shows only minimal toxic effects in previous toxicological studies.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, adrenals, bone (right hind leg), brain, eyes, heart, kidneys, liver, lungs, muscle (right hind leg), ovaries, renal fat, spleen, testes, thyroid and residual carcass. In addition, cage washes were collected.
Carcass, together with gastro-intestinal tract, was processed immediately after necropsy. All other samples (excreta, tissues and organs) were stored deep frozen until analysis.
- Time and frequency of sampling:
Urine: 6, 12, 24, and 48 h post dosing
Faeces: 24 and 48 h post dosing
After sample collection the metabolism cages were rinsed with water. These cage washes were kept separately.
Necropsy: 48 h post dosing
Blood (and plasma): taken at necropsy, 48 h post dosing

DETECTION METHOD:
Radioactivity was measured by liquid scintillation counting with automatic external standard quench correction. Corrected dpm of less than the background cpm were considered to be below the limit of quantification.

Statistics:

The concentration of tissue residues in all samples was calculated. Means and standard deviations for each analysis and for individual values in each group were calculated.

Type:

absorption

Results:

The minimum absorption of the test substance within 48 h as measured by urinary excretion did not differ between the sexes (61.8% in males and 63.3% in females) after oral administration of 457 mg/kg bw.

Type:

distribution

Results:

Sex differences were noted in the mean tissue residue values at necropsy with males showing generally higher residue levels than females. Radioactivity was mainly observed in the excretory organs kidney, liver and in skin (males) and renal fat (females).

Type:

excretion

Results:

Excretion of the administered dose was rapid in both sexes, however, females showed faster excretion rates than males. The excretion profile was similar between males and females.

Details on absorption:

The total amount of a dose that is excreted via the urine is a surrogate for the minimum amount that has been absorbed. Following the administration of a single dose of 457 mg/kg bw of the radiolabelled test substance, male and female urinary excretion after 48 h was 58.5 ± 12.9% and 59.9 ± 8.45% of administered dose, respectively. A further 3.28 ± 1.53% and 3.39 ± 1.71 % was recovered in cage washes over the experimental period for the male and female rats, respectively. Therefore, the minimum total absorption for male and female rats was 61.8 ± 12.5% and 63.3 ± 9.64%, respectively. This differs from the results seen after administration of the low dose, where only 45.9% of the dose was excreted in the urine by males, but 81.4% was excreted in the urine by females.

Details on distribution in tissues:

Differences in tissue distribution were noted between the sexes, with males generally showing higher residue levels than females. Highest residue levels in males were seen in the skin, kidney, and liver (15.5 ± 10.7, 10.1 ± 5.02, and 8.84 ± 4.12 mg equivalent test substance/kg tissue, respectively). The high levels in skin may be due to the test substance being associated with the subcutaneous fat or contamination of the fur by test substance-containing urine, while the tissues involved in the metabolism and elimination of the test substance (i.e. liver and kidney) were expected to show high residue levels. Residue levels in the heart of male rats were 5.81 ± 2.66 mg/kg tissue. With the exception of the carcass, all other residue levels were below 3 mg/kg tissue. The measured residue levels in the carcass (8.06 ± 4.20 mg/kg tissue) comprised residue levels in the gastro-intestinal tract and contamination of the fur by urine. This pattern of distribution in the male rat is similar to that noted after low dose administration, where the highest residue levels were found in the kidney, liver, heart, then skin.
In females, residue levels were highest in renal fat, liver, and kidney (5.50 ± 5.75, 4.81 ± 4.10, and 3.89 ± 2.86 mg/kg tissue, respectively). The measured residue levels in renal fat suggest a degree of distribution into fatty tissues, which is more extensive in females than males. The residue levels in skin for females were low relative to other tissues (0.602 ± 0.841 mg/kg tissue). As with males, the carcass also shows relatively high residue levels (3.92 ± 3.71 mg/kg tissue). The tissues with the highest residue levels measured in the present study are the same as those seen in the low dose study, albeit in a different order.
No residues were detectable in the spleen of both sexes. Lowest detectable residue levels in both sexes were in the eyes (0.997 ± 0.887 and 0.265 ± 0.376 mg/kg tissue, respectively for males and females) followed by testes for males (1.15 ± 0.450 mg/kg tissue) and muscle for females (0.443 ± 0.524 mg/kg tissue).

Details on excretion:

The excretion pattern was similar for both sexes. Elimination was via both urine and faeces (58.5 ± 12.9 and 34.4 ± 11.7% of administered dose for males, and 59.9 ± 8.45 and 34.9 ± 9.59% of administered dose for females, respectively). Females excreted the absorbed dose more rapidly than the males, with 42.9% of the total urinary excretion occurring in the first 6 h for female rats, compared to 20.7% for male rats. This was also reflected in faecal elimination, with 91.4% of faecal elimination occurring in the first 24 h for females, compared to 69.2% in males. These observations are in agreement with the residue data, where males showed higher tissue residue levels, and suggest that the test substance is less extensively distributed, and more rapidly eliminated in the female rat compared to the male. Total radioactivity recovered in the excreta by 48 h post dosing in males was 96.2 ± 2.71% and 98.2 ± 0.701% in females.
This data shows that the test substance at high dose is absorbed and eliminated rapidly by the rat. Although the elimination pathway is similar between the sexes, females are seen to excrete the compound more quickly.

Metabolites identified:

not measured

Table 1: Tissue distribution of radiolabelled residues following single oral dosing at 457 mg/kg bw

	Concentration (mg equivalent test substance/kg tissue)
	males		females
Tissue	Mean	SD	Mean	SD
Adrenals	1.380	1.300	0.965	1.170
Blood	1.480	1.010	0.407	0.687
Bone	2.250	0.958	0.863	0.860
Brain	2.400	2.280	0.847	0.622
Carcass	8.060	4.200	3.920	3.710
Eyes	0.997	0.887	0.265	0.376
Heart	5.810	2.660	2.080	2.520
Kidney	10.100	5.020	3.890	2.860
Liver	8.840	4.120	4.810	4.100
Lungs	1.980	1.350	0.928	0.995
Muscle	1.330	0.639	0.443	0.524
Ovaries	-	-	1.069	0.751
Plasma	2.260	1.630	0.390	0.872
Renal fat	2.630	1.630	5.500	5.750
Skin	15.500	10.700	0.729	0.772
Spleen	BLQ	-	BLQ	-
Testes	1.150	0.450	-	-
Thyroid	1.430	0.971	0.706	1.010

BLQ = Below level of quantification (mean dpm for sample is < twice background dpm).

Table 2: Elimination of radiolabelled material following single oral dosing at 457 mg/kg bw

		Percentage of radioactive dose
		Males		Females
		Mean	SD	Mean	SD
Urine	6h	12.10	3.29	25.70	8.51
	12h	11.10	2.71	11.20	3.62
	24h	20.80	6.73	14.40	4.32
	48h	14.50	9.25	8.59	13.10
	Sub-total	58.50	12.90	59.90	8.45
Faeces	24h	23.80	16.60	31.90	13.50
	48h	10.60	6.00	3.05	4.82
	Sub-total	34.40	11.70	34.90	9.59
Cage wash	24h	2.37	1.28	2.69	1.06
	48h	0.90	0.52	0.70	0.73
	Sub-total	3.28	1.53	3.39	1.71
% Total eliminated		96.20	2.71	98.23	0.70
% Carcass		1.61	0.82	0.76	0.71
% Total recovery		97.70	2.26	99.00	0.74

Conclusions:

Based on the results of the present study, the test substance was rapidly absorbed and eliminated by rats. A small, but significant, sex difference in the magnitude of the tissue residues and in the rate, but not route, of excretion was apparent. No bioaccumulation potential was observed after single administration.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Aug - 03 Sep 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

distribution

excretion

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

Adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Council Directive 91/414/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: J MAFF 59 NohSan No 4200

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C labelled test substance

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL: (IGS) CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, UK
- Body weight at receipt: 138 - 167 g (male and female)
- Fasting period before study: no
- Housing: During acclimatisation and during treatment with the radiolabelled test substance, the rats were housed in single sex groups of 3 rats in plastic holding cages with wire mesh and trays lined with paper underneath to catch the droppings.
- Individual metabolism cages: yes, only the rats used for the depuration study were transferred to individual all-glass metabolism cages, following the last of 14 consecutive daily doses with the radiolabelled test substance
- Diet: powdered laboratory rodent diet SDS RMI(E) SQC FG, except for the animals of the depuration study who received pelleted diet (Harlan Techlab) on transferral to metabolism cages, ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Aug 1998 To: 03 Sep 1998

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Two dosing solutions were prepared for the present study. Dose 1 was used from day 0 - 6 and dose 2 from day 7 - 14 of administration.
A co-precipitate was prepared by dissolving accurately weighed amounts of radiolabelled test substance or unlabelled test substance in chloroform. The chloroform was removed under a stream of nitrogen followed by desiccation for approximately 3 - 4 h.
Each dosing solution was prepared by dissolving accurately weighed amounts of the co-precipitate in sesame oil at a nominal concentration of 2 mg/mL. The actual concentration achieved was 1.996 mg/mL with a specific activity of 19.6517 µCi/mg (727.113 Mbq/g) for dose 1, and 1.994 mg/mL with a specific activity of 19.8490 µCi/mg (734.413 Mbq/g) for dose 2.

VEHICLE
- Justification for use and choice of vehicle: Sesame oil was used as preliminary toxicology studies were conducted using it as the dosing vehicle for gavage studies
- Concentration in vehicle: 2 mg/mL
- Amount of vehicle: 5 mL/kg bw

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
The radiochemical purity of each dosing solution was determined by HPLC. The results gave a purity of 99.63 ± 0.1% for dose 1 and 99.62 ± 0.1% for dose 2. The stability of the test substance in the vehicle was determined by analysis of aliquots of dose 1 and dose 2 by HPLC after 6 and 7 days, respectively. The radiochemical purity was 99.60 ± 0.1% for dose 1 over the 6 day period and 99.63 ± 0.0% for dose 2 over the 7 day period. The test substance in the dosing solution was therefore shown to be stable under the conditions of storage over the dosing period.

Duration and frequency of treatment / exposure:

group 1 (measurement of tissue residues): single treatment (Day 1)
group 2 (measurement of tissue residues): 5 consecutive days (Day 1 - 5)
group 3 (measurement of tissue residues): 10 consecutive days (Day 1 - 10)
group 4 (measurement of tissue residues): 14 consecutive days (Day 1 - 14)
group 5 (measurement of excreta and tissue residues): 14 consecutive days (Day 1 - 14)

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

actual dose received, group 1 - 4: males: 10.211 ± 0.319 mg/kg bw/day, females: 10.275 ± 0.359 mg/kg bw/day
actual dose received, group 5: males: 10.149 ± 0.370 mg/kg bw/day, females: 10.386 ± 0.391 mg/kg bw/day

No. of animals per sex per dose / concentration:

3 animals per sex per group

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (absorption, distribution, excretion)
- Tissues and body fluids sampled of all animals: blood, plasma, adrenals, bone, brain, eyes, heart, kidneys, liver, lungs, muscle, ovaries, renal fat, skin, spleen, testes, thyroid and carcass with gastrointestinal tract (GIT). If they were not processed immediately, all tissues were stored under deep freeze conditions until analysis.
- Tissues and body fluids sampled only of group 5 animals: urine, faeces. In addition, cage washes were collected.
- Time and frequency of sampling:
Examinations of tissue residues of group 1 - 4 animals: Groups of 3 males and 3 females were killed by aortic bleeding under deep isoflurane anaesthesia on days 2, 6, 11, and 15, 24 h after receiving 1, 5, 10, or 14 daily doses, respectively.
Examinations of excreta of group 5 animals: Following administration of the 14th consecutive dose, 3 males and 3 females were placed in metabolism cages (metabowls). Urine and faeces were collected separately over dry ice at the following times points after dosing: 6 and 12 h (urine only), 24 and 48 h. At the final collection point the metabolism cages were rinsed with water. The cage washes were analysed separately. At 2 days following dose administration (Day 16) the animals were sacrificed and tissues/organs sampled as described above.

DETECTION METHOD:
Radioactivity was measured by liquid scintillation counting with automatic external standard quench correction. Corrected dpm of less than the background cpm were considered to be below the limit of quantification.

Statistics:

Percentage dose present and concentration of radioactivity in all samples were calculated by the DEBRA database. Means and standard deviations for each analysis and for individual values in each group were calculated.

Type:

distribution

Results:

The results of the tissue residue examinations showed that the test substance displays little tendency to accumulate in tissues after repeated dosing for 14 days, and is rapidly cleared from the investigated organs following cessation of dosing.

Type:

excretion

Results:

After repeated exposure the test substance was rapidly and completely excreted with 78 - 86% being found in the 0 - 24 h excreta. There was no significant difference in the rate or route of excretion compared to a single dose.

Details on distribution in tissues:

The measurements of radiolabelled residues in the tissues of rats, 24 h after a single oral dose of 10 mg/kg bw, revealed higher residue concentrations in males than in females, but overall the residue concentrations were generally low. With the exception of the carcass with GlT, for males only the plasma, heart, liver, and kidney contained residues greater than 1 µg/g tissue (1.115, 2.848, 2.531, and 4.366 µg/g tissue, respectively). In the case of females, only the kidney (2.372 µg/g tissue) contained residues above 1 µg/g tissue, the remaining residues being below 0.2 µg/g tissue with the exception of the heart and liver (0.574 and 0.342 µg/g tissue, respectively).
At 24 h after the last of 14 daily doses the concentrations of the test substance residues had increased or remained at similar levels with repeated dosing for all tissues, though the extent of the increase was small and the overall concentration of the residues was still low. The levels of radiolabelled residues in the tissues of rats remained higher in males than in females with the exception of the renal fat (0.688 µg/g tissue in males and 1.379 µg/g tissue in females). In male rats only the skin, plasma, blood, heart, liver, kidney, and muscle contained residues greater than 1 µg/g tissue (1.261, 1.576, 1.022, 4.034, 5.316, 7.684, and 1.185 µg/g tissue, respectively) with the highest residues found in the liver and kidney. In the case of females, only the renal fat, liver, and kidney were found to contain residues greater than 1 µg/g tissue (1.379, 1.659, and 3.093 µg/g tissue, respectively) with the residue concentration still being below 0.2 µg/g tissue in the plasma, blood, eyes, brain, lungs, spleen, and bone.
Following the 2-day depuration phase, tissue residue concentrations fell considerably with the exception of the renal fat and liver in the case of female rats. There were also slight reported increases in levels of residues in the brain, thyroid, ovaries, and adrenals of females, though these findings may not be significant due to the very low concentrations of radioactivity in these tissues where small changes in the radioactivity counted will produce disproportionately large changes in the residues concentration. In most tissues the final concentrations were similar to, or less than, those seen in the Group 1 rats which were killed 24 h after a single oral dose.
The data show that for all tissues the concentration of the test substance residues increased with repeated dosing though the extent of the increase was small. In male rats, the concentration of residues in the skin, eyes, adrenals, and bone increased by 4.472, 5.581, 4.669, and 3.953-fold, respectively. The residue concentrations in the rest of the tissues increased by less than 2.5-fold. Following a second depuration day, the concentration of the residues in male tissue fell considerably, by 39 - 94%, to return to similar levels seen 24 h after a single dose. In the case of female rats, the largest increases in the concentration of residues were seen in the lipophilic tissues, in particular the renal fat, ovaries and thyroid, together with the skin, which also included some subcutaneous fat. For these tissues, the maximum concentration of residues was seen 48 h after the final dose though the magnitude of the residues was still small. For the majority of the tissues the residue concentrations increased by less than 2.5-fold over the 14 days of dosing and fell to levels close to those seen after a single dose following a 48 h depuration phase.
The highest residue concentrations were seen in the kidney, liver, and heart of both male and female rats and in the renal fat of female rats. In the remainder of the tissues, the concentration of the test substance residues remained low throughout the study, and peaked at 0.039 - 1.576 µg/g tissue in the tissues of male rats, and at 0.01 - 0.674 µg/g tissue in the case of female rats.
The results show that the concentrations of the test substance residues for all tissues (with the exception of renal fat) were higher in the male than in the female. The maximum mean concentration of radiolabelled residues was found to be 1.7 - 8.9 times greater in males than females. In renal fat the maximum mean concentration of radiolabelled residues were found to be 3 times greater in females than males.
The concentration of radiolabelled residues in blood and plasma of the rats peaked at values of 1.022 µg/g tissue and 1.576 µg/g tissue in the case of male rats, and 0.116 µg/g tissue and 0.178 µg/g tissue in the case of female rats, respectively. At the end of the depuration phase the concentration of radiolabelled residues in the blood were 84.3 and 57.8% of the maximum value. In plasma, the concentration had fallen to 94.0 and 75.8% of maximum residue levels for males and females, respectively.
Overall the results of the tissue residue study show that the test substance displays little tendency to accumulate in tissues with repeated dosing, and is rapidly cleared from the major organs following cessation of dosing.

Details on excretion:

The amount of radiolabelled residues excreted in the urine and faeces has been quantified against the total dose of radiolabelled test substance administered to the rats over the 14 days administration period. Because the excreta were only collected after administration of the final dose, the overall recovery was <90%. In order to compare the excretion profile with that found after a single dose, the excretion data have been normalised by converting the values to a percentage of the recovered radioactivity rather than the dosed radioactivity. This enables the rate and route of excretion to be more easily compared to the results previously obtained for the excretion following a single oral dose. The results show that the overall recovery, based on the total dose given, was 9.979 ± 0.382% in the case of male rats and 7.983 ± 0.148% in the case of female rats. In the case of tissues where the whole organ was not removed (e.g. skin, muscle etc.) a factor was used based on the % body weight, to enable radioactivity in the total tissue to be assessed. The tissues that contained the greatest amount of dosed radioactivity were found to be the renal fat, skin, and muscle, which contained the equivalent of 0.024 - 0.107%, 0.048 - 0.063% and 0.021 - 0.041%, respectively of the total dose administered. The liver and kidney contained the equivalent of 0.051 - 0.064% and. 0.012 - 0.017% respectively of the total dose administered. Normalisation of the excretion data revealed that the dose was very rapidly excreted with 78.5% of the recovered radioactivity being found in the 0 - 24 h excreta of males and 86.4% being found in the 0 - 24 h excreta of females. Urinary excretion predominated in female rats and faecal excretion in males with 38.525 ± 5.238% and 87.606 ± 1.849% of the excreted radioactivity found in the urine of male and female rats, respectively. These results are similar to those found after administration of a single oral dose, though the percentage excreted in the faeces by female rats was reduced. The highest concentrations of radioactivity were found in the kidney and liver of male rats with mean values in the kidney of 2.840 µg/g tissue and 2.184 µg/g tissue, respectively. The highest concentrations of radioactivity were found in the renal fat, kidney, and liver of female rats with 2.067 µg/g tissue, 1.946 µg/g tissue, and 1.726 µg/g tissue, respectively. All other tissues contained concentrations of residues below 1 µg/g tissue. Overall, the results for the excretion balance study indicated that the route and rate of excretion do not differ considerably after single or repeated exposure to the test substance.

Metabolites identified:

not measured

Table 1: Tissue distribution of radiolabelled test substance residues following repeated daily oral dosing of 10 mg/kg bw/day test substance for up to 14 days

	Mean tissue residue concentration (mg equivalent test substance/kg tissue)
	males					females
Study day (day of necropsy)	2	6	11	15	16	2	6	11	15	16
Carcass/GIT	1.911	1.599	2.598	3.168	0.739	0.551	0.867	0.987	1.399	0.689
Skin*	0.282	0.536	0.680	1.261	0.429	0.099	0.211	0.347	0.674	0.371
Plasma	1.115	0.781	1.109	1.576	0.095	0.178	0.063	0.096	0.095	0.043
Blood	0.679	0.516	0.735	1.022	0.160	0.116	0.048	0.075	0.085	0.049
Eyes	0.047	0.060	0.075	0.275	0.051	0.024	0.011	0.016	0.046	0.028
Brain	0.032	0.022	0.039	0.035	0.011	0.010	0.005	0.006	0.007	0.014
Renal fat	0.327	0.294	0.417	0.688	0.418	0.144	0.483	1.031	1.379	2.067
Heart	2.848	3.051	3.197	4.034	0.226	0.574	0.644	0.956	0.847	0.109
Lungs	0.812	0.534	0.865	0.966	0.091	0.123	0.079	0.103	0.140	0.058
Spleen	0.152	0.144	0.220	0.322	0.135	0.043	0.030	0.043	0.061	0.038
Liver	2.531	2.860	4.130	5.316	2.184	0.342	0.678	1.351	1.659	1.726
Kidney	4.366	5.792	5.224	7.684	2.840	2.372	2.235	2.490	3.093	1.946
Thyroid	0.327	0.432	0.238	0.755	0.298	0.037	0.081	0.100	0.255	0.275
Ovaries	-	-	-	-	-	0.050	0.104	0.199	0.334	0.407
Testes	0.473	0.399	0.496	0.803	0.150	-	-	-	-	-
Adrenal	0.169	0.141	0.237	0.789	0.444	0.068	0.100	0.153	0.344	0.461
Muscle	0.653	0.732	1.180	1.185	0.113	0.117	0.121	0.262	0.220	0.063
Bone	0.148	0.189	0.258	0.585	0.156	0.048	0.063	0.081	0.142	0.092

* These results may be affected by contamination from urine during housing.

Table 2: Mean concentrations of radiolabelled test substance residues in the tissues of rats following repeated daily oral dosing of 10 mg/kg bw/day and the degree of depuration following cessation of dosing

	Conc. 24 h after a single dose (Csingle) (µg/g tissue)*	Maximum residue tissue conc. (Cmax) (µg/g tissue)	Ratio Cmax/Csingle	Conc. 48 h after last dosing (µg/g tissue)**	% decrease from Cmax after 48 h depuration	Conc. 24 h after a single dose (Csingle) (µg/g tissue)*	Maximum residue tissue conc. (Cmax) (µg/g tissue)	Ratio Cmax/Csingle	Conc. 48 h after last dosing (µg/g tissue)**	% decrease from Cmax after 48 h depuration
	male					female
Skin***	0.282	1.261	4.472	0.429	66.0	0.099	0.674	6.808	0.371	45.0
Plasma	1.115	1.576	1.413	0.095	94.0	0.178	0.178	1.000	0.043	75.8
Blood	0.679	1.022	1.505	0.160	84.3	0.116	0.116	1.000	0.049	57.8
Eyes	0.047	0.275	5.851	0.051	81.5	0.024	0.046	1.917	0.028	39.1
Brain	0.032	0.039	1.219	0.011	71.8	0.010	0.014	1.400	0.014	0.0
Renal fat	0.327	0.688	2.104	0.418	39.2	0.144	2.067	14.354	2.067	0.0
Heart	2.848	4.034	1.416	0.226	94.4	0.574	0.956	1.666	0.109	88.6
Lungs	0.812	0.966	1.190	0.091	90.6	0.123	0.140	1.138	0.058	58.6
Spleen	0.152	0.322	2.118	0.135	58.1	0.043	0.061	1.419	0.038	37.7
Liver	2.531	5.316	2.100	2.184	58.9	0.342	1.726	5.047	1.726	0.0
Kidney	4.366	7.684	1.760	2.840	63.0	2.372	3.093	1.304	1.946	37.1
Thyroid	0.327	0.755	2.309	0.298	60.5	0.037	0.275	7.432	0.275	0.0
Testes	0.473	0.803	1.698	0.150	81.3	-	-	-	-	-
Ovaries	-	-	-	-	-	0.050	0.407	8.140	0.407	0.0
Adrenal	0.169	0.789	4.669	0.444	43.7	0.068	0.461	6.779	0.461	0.0
Muscle	0.653	1.185	1.815	0.113	90.5	0.117	0.262	2.239	0.063	76.0
Bone	0.148	0.585	3.953	0.156	73.3	0.048	0.142	2.958	0.092	35.2

* Mean residue concentration of Group 1

** Mean residue concentration of Group 5

*** These results may be affected by contamination from urine during housing.

Table 3: Elimination of radiolabelled test substance following 14 daily oral doses of 10 mg/kg bw/day

		Cumulative excretion of radioactivity (expressed as % of total dose)
		Males		Females
Tissue	Time point	Mean	SD	Mean	SD
Urine	6h	0.482	0.309	3.043	1.568
	12h	1.986	0.404	5.700	0.607
	24h	3.391	0.661	6.494	0.365
	48h	3.800	0.661	6.834	0.237
Faeces	24h	4.337	0.197	0.246	0.022
	48h	5.299	0.450	0.319	0.012
Cage wash	48h	0.144	0.062	0.147	0.133
Sub-total		9.243	0.339	7.300	0.132
Carcass/GIT		0.505	0.056	0.433	0.050
Other tissues*		0.230	0.027	0.250	0.046
% Total recovery		9.979	0.382	7.983	0.148

* These values were calculated using total tissue weights obtained from literature

Conclusions:

Following repeated daily dosing for 14 days, the concentration of radioactive residues in the tissues increased minimally. 48 h after the final dose, the concentration of radiolabelled residues in most tissues decreased to levels close to those found 24 h after a single dose, demonstrating the rapid clearance of radiolabelled residues.
The magnitude of radioactive residues was greater in males than females for all tissues examined, except for renal fat.
The 48 h-excretion profile seen subsequently to 14 daily doses showed that the applied dose was rapidly and completely excreted with 78 - 86% being found in the 0 - 24 h excreta. Whereas the repeated daily dosing had no significant effect on the rate or route of excretion of the test substance compared to single dosing.
No bioaccumulation potential was observed under the conditions of the present study.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 May 1996 - 3 Sep 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

Adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC: 94/97/EC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan: J-MAFF

Deviations:

no

Principles of method if other than guideline:

The present study was conducted to determine the metabolism of the test substance in the rat following oral administration of a single dose of 10 mg/kg bw (study number M-142588-01-1), 457 mg/kg bw (study number M-142589-01-1), or following repeated oral dosing for 14 days at 10 mg/kg bw/day (study number M-183660-01-1).

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C labelled test substance

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, UK
- Age at study initiation: young adult
- Body weight range at start of dosing: 151 - 229 g (not further specified)
- Housing: prior to dosing, rats were housed in single sex groups in plastic cages with wire mesh and paper trays underneath to catch the droppings
- Individual metabolism cages: yes, following dosing, the male and female animals were housed individually in all-glass metabolism cages, which were equipped to collect urine and faeces under cooled conditions to prevent bacterial degradation of the metabolic products.
- Diet: powdered laboratory rodent diet SDS-RMI(E)SQCFGPL under normal holding conditions, pellet diet Harlan Teklad TRM during the time in the metabolism cages, always ad libitum
- Water: tap water in drinking water quality, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
low dose study: From: 21 May 1996 To: 23 May 1996
high dose study: From: 09 Jul 1996 To: 11 Jul 1996
repeated dose study: From: 19 Aug 1998 To: 03 Sep 1998

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The radiolabelled test substance or the unlabelled test substance were either directly dissolved in sesame oil for preparation of low dose-dosing solutions, or, for high dose- and repeated dose-dosing solutions, the radiolabelled/unlabelled test substance was first dissolved in chloroform, blown to dryness under nitrogen, desiccated, and then resuspended in sesame oil.

Duration and frequency of treatment / exposure:

single treatment or repeated daily treatment for 14 consecutive days

Dose / conc.:

10 mg/kg bw (total dose)

Remarks:

single treatment, low dose

Dose / conc.:

457 mg/kg bw (total dose)

Remarks:

single treatment, high dose

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

repeated dosing for up to 14 days

No. of animals per sex per dose / concentration:

single treatment: 5 animals per sex
repeated treatment: 3 animals per sex per group

Control animals:

no

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces. In addition, cage washes were collected.
- Time and frequency of sampling: 6, 12, 24, and 48 h post dosing
- From how many animals: Samples of urine were pooled in proportion to their total volume to form one representative sample for males and females, respectively, for each collection time point from rats dosed once at either 10 or 457 mg/kg bw. For the repeated dose study, the metabolic profile of individual urine samples was determined. Faeces samples were also pooled to form one representative sample for males and females, respectively, for each collection time point.
- Method type(s) for identification: liquid scintillation counting, HPLC-UV, mass spectrometry, NMR, TLC

Statistics:

The amount of each radioactive entity present in the samples analysed by HPLC was determined as a percentage of the total radioactivity in the sample by the Labchrom program or the Reeve 27000 integration system. These values were then converted manually to a percentage of the original dose given to the animals. Due to the number of significant figures that the TRACE and DEBRA programs work to, calculated figures may differ from manually calculated figures. From the TRACE manual, the acceptable criterion for % values is ± 1.0%.

Type:

metabolism

Results:

amounts of metabolites excreted via urine and faeces after single low dose (10 mg/kg bw): males: 31.55% free acid, 31.89% oxidised metabolite, 0.20% test substance, females: 83.30% free acid, 6.01% oxidised metabolite, 0.06% test substance

Type:

metabolism

Results:

amounts of metabolites excreted via urine and faeces after single high dose (457 mg/kg bw): males: 70.83% free acid, 13.86% oxidised metabolite, 0.24% test substance, females: 79.18% free acid, 4.40% oxidised metabolite, 0.0% test substance

Type:

metabolism

Results:

amounts of metabolites excreted via urine and faeces after repeated dose (10 mg/kg bw/day for 14 days): males: 31.10% free acid, 43.81% oxidised metabolite, 0.48% test substance, females: 84.40% free acid, 5.08% oxidised metabolite, 0.11% test substance

Metabolites identified:

yes

Details on metabolites:

Identification of urinary metabolites:
In a preliminary toxicokinetics study (study number M-139553-01-1), analysis of urine samples by HPLC showed the presence of two major metabolites that appeared to be of greater polarity than the test substance (parent compound). One of the metabolites had the same retention time on HPLC, and the same Rf on TLC, as the free acid. The second metabolite was more polar in nature and was not characterised further. In order to obtain sufficient amounts of material for the isolation and identification of the urinary metabolites for the present study, additional male rats were dosed at 75 mg test substance/kg bw. The HPLC profiles of the urine samples were similar to those from the urines obtained in the preliminary study. Male rats were used since the early data indicated that they produced more of the more polar major metabolite than did the females.
The individual metabolites were isolated by HPLC of pooled urine samples followed by collection of the radiopeaks. The major peak had been shown in the preliminary study to co-chromatograph with the free acid (5,5-diphenyl-2-isoxazoline-3-carboxylic acid). This metabolite would be readily formed from the test substance by ester hydrolysis. The structure of this metabolite was confirmed by mass spectrometry. The second metabolite was a major component in the urine of male rats, but was only a minor component in the urine of female rats. The peak was isolated by HPLC and concentrated to dryness for analysis by NMR. The NMR data identified this peak as a para-substituted hydroxyacid. This metabolite was shown to have the same retention time as the synthetic standard 5-(4-hydroxyphenyl)-5-phenyl-2-isoxazoline-3-carboxylic acid on HPLC. This result was further confirmed by mass spectrometry.
A number of minor polar metabolites appeared in the radiotrace of the HPLC. They were present in amounts < 3% of the applied dose with the majority being < 1% of the applied dose. These metabolites were not further characterized.

Identification of faecal metabolites:
Initial analysis of faecal homogenate indicated the presence of several minor metabolites, in addition to the parent compound and a major metabolite with similar retention time to the free acid-metabolite. In the present study, faeces were analysed using samples from male rats dosed at 75 mg/kg bw. Analysis of the reduced aqueous, NaOH and methanol extracts indicated a similar metabolite profile to that seen in urine with the addition of the presence of small amounts (< 0.5%) of the parent compound. A second metabolite, which co-chromatographed with the free acid-metabolite and a third metabolite with a similar retention to the hydroxyacid-metabolite were also present. In addition, several minor (3% of the applied dose) polar metabolites were noted in the aqueous extract, without further examination.

Identification of plant metabolites as metabolites of the test substance (parent compound) in the rat:
In a separate experiment, the presence of three putative plant metabolites in the excreta of male rats was determined. Three putative plant metabolites (3-Hydroxy-3,3-diphenylpropanenitrile; 3-Hydroxy-3,3-diphenylpropanol oxime; 3-Hydroxy-3,3-diphenylpropanoic acid) were run on HPLC, to determine whether they were present as minor metabolites in the excreta of treated rats. The metabolites were not detected in urine, but analysis of the methanol extracts of faeces identified the presence of trace amounts (< 0.5% of the applied dose) of 3-Hydroxy-3,3-diphenylpropanenitrile by co-chromatography with the synthetic standard. The other two compounds were not detected.

Quantification of metabolites of the test substance (parent compound) in urine and faeces:
Aliquots of the urine samples pooled within sexes for each collection time-point from the rats dosed with a single oral dose of 10 or 457 mg radiolabelled test substance/kg bw, or following 14 daily doses of 10 mg/kg bw/day, were applied to an HPLC column. The resulting radiotrace was integrated to determine the contribution of each of the metabolites of the test substance present and expressed as % of radioactivity analysed. This value was then used, together with the amount of dose present in the excreta sample, to quantify the amount of each of the metabolites present as a % of dosed radioactivity.
In addition to the radioactive metabolite peaks that corresponded to the parent substance, the free acid-metabolite, and the hydroxyacid-metabolite, several other minor peaks were detected on the radiotrace throughout the HPLC run. Many of these peaks were not consistently present in all samples, their amount accounted for < 1% of the applied dose (following integration), and thus, they were not further characterized.
The results of the analyses confirmed that there was a significant sex difference in the metabolism of the test substance in rats. In females, ester hydrolysis to produce the free acid-metabolite was the major route accounting for 79 - 84% of the dose. The hydroxyacid-metabolite was present in small amounts accounting for 4.4 - 6% of the dose at all dose levels. In males, the metabolic profile changed after repeated dosing. At the single low dose of 10 mg/kg bw the amount of the free acid- and the hydroxyacid-metabolite present in the excreta was approximately equal (31.9% and 31.6% of the applied dose, respectively). The formation of the hydroxyacid-metabolite increased following repeated dosing (43.8% of the applied dose), whereas the amount of the free acid did not change (31.1% of the applied dose). At the single high dose of 457 mg/kg bw however, the metabolic pathway leading to the formation of the hydroxyacid-metabolite appeared to be saturated, as the free acid became the major metabolic product (13.9% and 70.83% of the applied dose, respectively).

SUMMARY - Main metabolites found in urine and faeces:
The main metabolite was identified by HPLC (co-chromatograph) and mass spectrometry as the free acid of the test substance (ester hydrolysis resulting in the free acid). The free acid was identified as the major metabolite in the excreta of female rats at all dosing regimes, and of male rats at the high dose.
The second metabolite, which was more polar than the free acid, was identified by HPLC and NMR. The results of the analysis suggested that this metabolite was oxidised in the 4-position of the phenyl ring (para-substituted hydroxyacid). The structure of the metabolite was confirmed by mass spectrometry. It was a major metabolite in male rats at the low dose (single and repeated) but this metabolic route appeared to become saturated at the high dose. The sex difference observed in the formation of the hydroxyacid-metabolite could explain the differences seen in the excretion profile and pharmacokinetics of the compound.

Table 1: Amounts of each metabolite of the test substance excreted in the urine and faeces of rats following oral dosing

		% of administered oral dose
		Free acid	Hydroxyacid metabolite	Test substance (parent compound)	Total
Single low dose 10 mg/kg bw	Male	31.55	31.89	0.20	63.64
	Female	83.297	6.005	0.061	89.363
Single high dose 457 mg/kg bw	Male	70.83	13.86	0.24	84.93
	Female	79.18	4.40	0	83.58
Repeated dose 14 days 10 mg/kg bw/day	Male	31.10	43.81	0.48	75.39
	Female	84.40	5.083	0.111	89.594

Conclusions:

Based on the results of the present study, the test substance was almost completely metabolised following oral absorption by the rat. Unchanged test substance was observed only in trace amounts (< 0.5%) in the excreta of treated animals.
The major route of metabolism was hydrolysis of the ester to yield the free acid.
A second metabolite, formed by oxidation on the phenyl ring, was another main metabolite in male animals.
No bioaccumulation potential was observed.

Description of key information

The test substance is readily absorbed after oral exposure and systemically available, with highest tissue residue levels in the liver and kidneys. The main metabolite was identified as the free acid. Excretion occurs via urine and faeces. No bioaccumulating potential was observed.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

The toxicokinetic behaviour of the test substance in male and female rats was investigated in several reliable studies where [14C]-radiolabelled test substance was applied via oral gavage. First results on absorption, distribution, metabolism and excretion were obtained in a preliminary study (M-139553-01-1, 1995). Subsequently, these were confirmed in two single dose studies (10 and 457 mg/kg bw) (M-142588-01-1, 1997, M-142589-01-1, 1997). Possible changes in metabolism and clearance after repeated exposure were assessed in an additional repeated dose study (10 mg/kg bw/day for 14 days) (M-183660-01-1, 1999). The analysis of urinary and faecal metabolites resulting from the described test regimes was reported in a separate study (M-184537-01-1, 1999).

 

Methods:

Single dose oral studies in rats:

Rats of both sexes received a single oral dose of [14C]-radiolabelled test substance at 10 and 500 mg/kg bw (M-139553-01-1). Animals were sacrificed 96 h post dosing and residue levels of radiolabelled material in the tissues were determined. After quantification of blood levels in these animals, further animals (1 per dose) were dosed at nominal doses of 5, 10, and 200 mg/kg bw. These animals were killed 1 or 4 h post dosing (tmax), when blood levels were expected to be at a maximum (Cmax). Levels of [14C] and unchanged test substance in these animals were determined. Subsequent definite single dose studies examining and determining the absorption, distribution and elimination of radiolabelled residues in the rat (5 animals/sex/dose) following a single oral dose with [14C]-radiolabelled test substance at 10 mg/kg bw and 457 mg/kg bw were conducted. Blood samples were frequently taken and excreta were collected. Animals were sacrificed 48 h post dosing and residue levels of radiolabelled material in the tissues were determined (M-142588-01-1, M-142589-01-1).

Repeated dose oral study in the rat:

In a repeated dose oral study male and female rats were given a single daily oral dose of 10 mg/kg bw test substance for up to 14 days (M-183660-01-1). Rats (3 animals/sex/time point) were sacrificed 24 h after having received 1, 5, 10, or 14 doses. At necropsy whole tissues or representative samples were removed and analysed to determine the content of radioactive residues. The rats treated for 14 days (3 animals/sex) were placed into metabolism cages. Urine and faeces were collected over the next 48 h. At necropsy whole tissues or representative samples were removed and analysed by scintillation counting.

  

ABSORPTION

Oral:

Absorption following dosing at 10 mg/kg bw was quicker in the females (1.0 h to reach maximum blood concentration) compared to males (4.0 h). After dosing with 500 mg/kg bw, absorption was slowed such that maximum blood concentration was not reached until 4.0 h (male and female). Blood concentrations of radiolabelled material declined biphasically, with terminal half-lives of 38.9 h (male) and 32.3 h (female) from animals dosed at 10 mg/kg bw and 14.4 h (male) and 30.1 h (female) from animals dosed at 500 mg/kg bw. The female low dose blood concentration/time curves showed a far higher clearance than the male low dose animals (approximately 8-fold difference) and a reduced AUC (approximately 7-fold lower). In the high dose animals, these sex differences were not seen, as the clearance and AUC figures were similar for both sexes (M-139553-01-1).

In the low dose group of the definite study minimum absorption of the test substance as measured by urinary excretion differed between the sexes (45.9% in males and 81.4% in females). This sex difference was no longer observed in the high dose group (61.8% in males and 63.3% in females). These values correspond to the minimum amount absorbed, since biliary excretion of the compound may have occurred but was not investigated (M-142588-01-1, M-142589-01-1).

In a repeated dose oral study, the concentration of residues in the tissues was generally below 1 µg/g tissue at 24 h after a single oral dose of 10 mg/kg bw. Following repeated daily dosing the concentration of radioactive residues in the tissues rose, though the extent of the increase was small (<2.5 for the majority of the tissues) and the overall magnitude of the residues remained low. At 48 h after the final dose, the concentration of residues in most of the tissues had fallen to levels close to those found 24 h after a single dose, demonstrating the rapid clearance of radioactivity (M-183660-01-1).

Overall the results of these studies in rats showed that after oral administration, the test substance was absorbed at a high rate and rapidly eliminated.

 

Dermal:

Dermal absorption of the test substance is expected to be very low (0.00002 mg/cm²/h; DERMWIN) based on a water solubility of 1.06 mg/L at 20 °C and a log Pow of 3.8. In addition, acute and repeated dermal application did not reveal any systemic effects up to the highest dose tested, whereas toxicity was observed after acute and repeated oral application, showing that dermal absorption is lower than oral (M-133573-01-1, M-133138-01-1, M-191414-01-1, M-184633-01-1).

Skin sensitization was observed in a guinea pig maximization test, showing that skin penetration does occur to a certain extent (M-141891-01-1).

 

Inhalation:

The vapour pressure of the test substance (2.2 E-06 Pa) indicates that inhalation of the test substance as a vapour is unlikely. The water solubility (1.06 mg/L) and log Pow of 3.8 suggest that absorption from the respiratory tract epithelium by passive diffusion is likely. As the substance is a solid, dust particles might be inhaled depending on the size (MMAD <100 µm). Particles (MMAD >15 µm) deposited on the mucociliary blanket will be elevated into the laryngeal region and ultimately be swallowed (ingestion). An acute inhalation toxicity study with the test substance showed no signs of systemic toxicity at 5040 mg/m³, indicating that the test substance might have a lower potential to be absorbed by the inhalation route than by the oral route (M-147615-01-1).

 

DISTRIBUTION

In the preliminary toxicokinetic study, tissue residues were mainly below the level of quantification in the low dose animals 96 h post dosing. The tissues with detectable levels of residues were the liver (0.112 and 0.037 µg/g tissue for males and females, respectively) and kidneys (0.359 and 0.131 µg/g tissue for males and females, respectively). Males had a 3-fold greater concentration of residues than females. In the high dose animals, residues were found in a greater number of tissues, but still at very low levels. Again the excretory organs liver (2.281 and 3.806 µg/g tissue for males and females, respectively) and kidneys (1.997 and 2.364 µg/g tissue for males and females, respectively) showed the highest concentrations of residues, but residues were also detected in renal fat (1.050 and 2.086 µg/g tissue, males and females, respectively) (M-139553-01-1).

 

In the two definite single dose studies sex differences were noted in the tissue residues at necropsy at both dose levels, with males showing generally higher residue levels than females. In the low dose group the highest residue levels in males were found in the excretory organs kidneys (0.71 mg equivalents/kg tissue) and liver (0.31 mg/kg tissue). In low dose group females, the highest residue levels were found in the kidneys (0.57 mg/kg tissue), renal fat (0.20 mg/kg tissue) and liver (0.16 mg/kg tissue). After treatment with 457 mg/kg bw test substance, the highest residue levels in males were found in the skin (15.5 ± 10.7 mg equivalents/kg tissue), kidney (10.1 ± 5.02 mg/kg tissue) and liver (8.84 ± 4.12 mg/kg tissue). In high dose group females, the highest residue levels were found in the renal fat (5.50 ± 5.75 mg/kg tissue), kidneys (3.89 ± 2.86 mg/kg tissue) and liver (4.81 ± 4.10 mg/kg tissue). Residues in the spleen were below the limit of quantification for both sexes in both groups, whereas residues in the brain and thyroid were only below the limit of quantification for both sexes in the low dose group (M-142588-01-1, M-142589-01-1).

In the repeated dose oral study the concentration of residues in the tissues was generally below 1 µg/g tissue 24 h after a single oral dose of 10 mg/kg bw test substance. Following repeated daily dosing the concentration of radioactive residues in the tissues rose, though the extent of the increase was small (< 2.5 for the majority of the tissues) and the overall magnitude of the residues remained low. At 48 h after the final dose, the concentration of residues in most of the tissues had fallen to levels close to those found 24 h after a single dose, demonstrating the rapid clearance of radioactivity. The magnitude of radioactive residues was greater in males than females for all tissues, except for renal fat (M-183660-01-1).

 

METABOLISM

Urine and faeces from the rats dosed with ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate (CAS No. 163520-33-0) was collected and metabolic profile of the compound was determined by HPLC. The metabolites were isolated from urine following solid phase extraction and HPLC, and the structures were determined by mass spectrometry and NMR. The metabolites present in the faeces were isolated by sequential extraction and HPLC. The structures of these metabolites were confirmed by co-chromatography with synthetic standards (M-184537-01-1).

The test substance was almost completely metabolised following oral absorption by the rat. Unchanged test substance was observed only in trace amounts (<0.5%) in the faeces and in low dosed male rat urine. The major route of metabolism was hydrolysis of the ester to yield the free acid 5,5-diphenyl-2-isoxazoline-3-carboxylic acid. This was the major metabolite in the female rat and in the male rat at the high dose level. A second metabolite 5-(4-hydroxyphenyl)-5-phenyl-2-isoxazoline-3-carboxylic acid, formed by oxidation on the phenyl ring, was a major metabolite in the male rat at the low dose only. This compound was a minor metabolite in the female rat and the male rat at the high dose. Repeated dosing with the test substance at 10 mg/kg bw/day had little effect on the metabolic profile of the compound in female rats, but increased the formation of 5-(4-hydroxyphenyl)-5-phenyl-2-isoxazoline-3-carboxylic acid in male rats. 3-Hydroxy-3,3-diphenylpropanenitrile, a plant metabolite, was observed as a trace metabolite of the test substance excreted in the faeces of the rat.

 

EXCRETION

The excretion profile of the [14C]-radiolabelled test substance differed between low and high dose groups. Low dose males excreted less in the urine and more in the faeces after 48 h (46% and 47%, respectively) than the low dose females, where 81% was recovered in urine and 11% in faeces 48 h post dosing. The sex difference was no longer evident in high dose males and females, where the excretion profile of the test substance was similar; with urinary excretion (not including cage washes) accounting for 59% (males) and 60% (females) and faecal elimination accounted for 34% (males) and 35% (females) of the administered dose. Excretion of the administered low and high dose was rapid in both sexes. However, females showed faster excretion rates than males in the high dose group. In the first 6 h 26% of the high dose had been excreted via the urine in females compared to 12% in males. By 48 h excretion levels were comparable in both dose groups. Overall recovery was 94 ± 2.36% and 93.51 ± 1.97% for males and females of the low dose group and 97.7 ± 2.26% and 99.0 ± 0.74% for males and females of the high dose group, respectively (M-142588-01-1, M-142589-01-1).

The excretion profile seen after 14 daily doses showed that the dose was rapidly and completely excreted with 78 - 86% being found in the 0 - 24 h excreta. The repeated daily dosing appeared to have no significant difference to the rate or route of excretion of the test substance.

 

CONCLUSION

After oral administration (10 or 500 mg/kg bw), the test substance was extensively metabolised, and only trace amounts of parent compound were detectable in excreta. The major metabolite at the high dose was identified as the free acid derivative of the parent ester. At the low dose, there was a marked quantitative sex difference in the metabolism profile. In females, the profile was similar to that seen at the high dose (mainly the acid metabolite) but in males a more polar metabolite, identified as the hydroxyl acid metabolite 5-(4-hydroxyphenyl)-5-phenyl-2-isoxazoline-3-carboxylic acid, was present. Repeated low dose exposure did not influence the metabolite profile in female rats but increased the formation of the hydroxyl acid metabolite in male rats. The sex difference in the metabolite profile seen in male animals of the low dose groups (single and repeated exposure) may account for the faster initial half-life in the blood, and the greater clearance seen in those animals. Urinary excretion was the major route of elimination, except in the low dose males where equal amounts were excreted in the faeces and urine. Elimination of radiolabelled test substance was rapid, with >90% of the dose excreted in either faeces or urine after 48 h in the low and high dose animals. In the low dose group but not the high dose group there was a sex difference in the magnitude of the tissue residues and in the route of excretion. Though the magnitude of the residues in the tissues increased with repeated dosing, the concentration of the residues remained low and the residues were rapidly cleared from the major organs following cessation of dosing.

Based on the available studies the test substance displays no bioaccumulation potential.