Chlorothalonil

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:

12 Oct 2016 to 23 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

excretion

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

other: EC 1107/2009

Version / remarks:

2009

Qualifier:

according to guideline

Guideline:

other: EU 283/2013

Version / remarks:

2013

Qualifier:

according to guideline

Guideline:

other: MAFF 12 Nohsan No 8147

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Remarks:

Han Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Groups 1& 3: 9-10 weeks; Groups 2 & 4: 8-9 weeks
- Weight at study initiation: 263-298 g for males; 154-190 g for females (Group 1); 236-266 g for males; 158-194 g for females (Group 2); 268-293 g for males; 174-188 g for females (Group 3); 259-298 g for males; 177-200 g for females (Group 4)
- Housing: Pre-study: Multiply housed by sex in solid bottomed polycarbonate cages with bedding. On study: Singly in all-glass metabolism cages (excretion Groups), multiply housed by sex in solid bottomed polycarbonate cages with raised wire mesh floors (kinetic Groups).
- Diet: A standard laboratory diet of known formulation available ad libitum
- Water: tap water ad libitum
- Acclimation period: At least 5 days prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 42 - 58
- Air changes: Minimum of 10/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 Oct 2016 To: 23 Jan 2017

Route of administration:

other: oral gavage and intravenous

Vehicle:

other: Oral: CMC, 0.5% aqueous solution; Intravenous: DMA: 20% Intralipid (5:95)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
See "other information on materials and methods incl. tables"

Duration and frequency of treatment / exposure:

Single oral dose administration (Group 1, excretion rats & Group 3, pharmacokinetic rats) or single intravenous dose administration (Group 2, excretion rats & Group 4, pharmacokinetic rats)

Dose / conc.:

1.5 mg/kg bw (total dose)

Remarks:

Group 1 & 3: Oral, no-effect dose

Dose / conc.:

0.5 mg/kg bw (total dose)

Remarks:

Group 2 & 4: Intravenous, reference route to interpret the level of absorption

No. of animals per sex per dose / concentration:

Four rats per sex per dose group

Control animals:

no

Details on study design:

A group of 4 male and 4 female rats per dose were each given either a single oral administration of nominally 1.5 mg/kg (Group 1, excretion rats & Group 3, pharmacokinetic rats) or a single intravenous administration of 0.5 mg/kg (Group 2, excretion rats & Group 4, pharmacokinetic rats) of [14C]-test substance. In excretion groups excreta samples were taken over predetermined time intervals up to 7 days post dose (Groups 1 and 2). In pharmacokinetic groups blood samples were taken over a 3 day period to determine the pharmacokinetics of total radioactivity in blood.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY
Excretion: Following dosing, urine and faeces were frozen upon excretion by collection over solid carbon dioxide (up to 168 hours post dose). Urine was collected predose and at 8 hours post dose, faeces was collected at predose, and urine and faeces were then collected at daily intervals until termination (168 hours post dose). The cages were rinsed with ca 5 mL of water prior to the removal of the urine pot. At each urine collection timepoint the cages were rinsed with a suitable volume of water and the washes collected separately. The concentration of total radioactivity was determined in each sample collected.
For rats used to collect excreta, the carcass was retained.

Pharmacokinetics: Blood samples (ca 0.1 mL) were collected from the jugular vein of each rat (into blood tubes containing lithium heparin anticoagulant) at a defined interval over a 72 hour period following dosing. The concentration of total radioactivity was determined in each blood sample collected.

Statistics:

Excretion: calculated mean and standard deviation
Pharmacokinetics: non-compartmental analysis

Type:

absorption

Results:

Based on urinary elimination the oral systemically available fraction was 18% in males and 19% in females

Type:

excretion

Results:

after a single oral dose of 1.5 mg/kg - faeces: 81 and 92% of the administered radioactivity in males and females, respectively; - urine: 5.4 and 6.4% of the dose in males and females, respectively; - cage wash: 0.6 and 1.5% for males and females

Type:

excretion

Results:

after a single intravenous dose of 0.5 mg/kg - faeces: 51 and 46% of the administered radioactivity in males and females, respectively; - urine: 36 and 39% of the dose in males and females, respectively; - cage wash: 2.4 and 4.1% for males and females

Details on absorption:

Following a single oral and intravenous administration of [14C]-test substance, the data suggest that differences exist between the fate of an intravenous dose and an oral dose. This is demonstrated in the underestimation of oral bioavailability (ca 2%) from the blood data, whereas, at least 5% of the oral dose was excreted in urine. This underestimation could be potentially associated with first pass metabolism, enterohepatic recirculation and/or possibly the reactive nature of the compound in blood and/or tissues.
Consequently, it was evident that the intravenous blood concentrations of radioactivity remained high throughout the study affecting the bioavailability estimate. In addition, at termination there is still ca 11% of the dose remaining in the carcass of the intravenous dose group animals.
The fraction of the oral dose systemically available, estimated by comparing the urinary excretion with that excreted following iv administration, was 18-19% of the administered dose.

Details on excretion:

Single oral dose (1.5 mg/kg bw): The majority of the administered radioactivity was excreted by 24 h post dose (82 and 91% in males and females, respectively). Excretion was essentially complete by 168 h post dose with 0.2% remaining in carcass in both sexes. The total mean recovery of administered radioactivity
including excreta, cage wash and residual carcass was 87 and 100% in male and female rats, respectively.

Single intravenous dose (0.5 mg/kg bw): The majority of the administered radioactivity was excreted by 24 h post dose (72% in both sexes). Excretion was not complete by 168 h post dose with 11-12% remaining in carcass. The total mean recovery of administered radioactivity including excreta, cage wash and residual
carcass was 101 and 102% in male and female rats, respectively.

Test no.:

#1

Toxicokinetic parameters:

Cmax: Two hours post dose in males (0.181 µg equiv/g) and 4 hours in females (0.118 µg equiv/g)

Remarks:

Single oral dose of 1.5 mg/kg bw

Test no.:

#2

Toxicokinetic parameters:

C(time): C0: 2.43 and 2.56 µg equiv/g, in males and females, respectively

Remarks:

Single intravenous dose of 0.5 mg/kg bw

Metabolites identified:

not measured

Analysis of dose preparation

Prior to dose preparation, the radiochemical was shown to have a purity of >97%. The pre and post-dose radiochemical purity of [14C]-test substance was >96% indicating that the test item was stable during dose preparation and for the duration of the dosing procedure. Analyses of individual aliquots of the oral dose preparation (Groups 1 and 3) prior to dosing were within 5.2% of the mean and therefore dosing proceeded. However analysis of individual aliquots taken throughout the dosing period were within 14.6% of the mean. The individual doses received by these animals were within an acceptable target range to allow the determination of absorption. Analyses of individual aliquots of the intravenous dose preparation (Groups 2 and 4) were within 4.2% of the mean, indicating that a satisfactory homogeneity had been achieved. The group mean achieved oral doses for [14C]-test substance were 1.15 and 1.15 mg/kg (Group 1) and 1.16 and 1.17 mg/kg (Group 3) for males and females. The group mean achieved intravenous doses for [14C]-test substance were 0.469 and 0.443 mg/kg (Group 2) and 0.453 and 0.449 mg/kg (Group 4) for males and females. 

Table 1. Mean Excretion of Radioactivity (as Percentage of Administered Dose) Following a Single Oral (1.5 mg/kg) or Intravenous (0.5 mg/kg) Dose of [14C]-test substance

Time after dosing (h)	Group 1 - 1.5 mg/kg (oral)		Group 2 - 0.5 mg/kg (iv)
	Male (n=4)	Female (n=4)	Male (n=4)	Female (n=3)
Urine
0-8	4.6	4.8	28	29
8-24	0.7	1.4	3.7	5.0
24-48	0.1	0.2	1.9	1.9
48-72	<0.1	<0.1	1.1	1.1
72-96	<0.1	<0.1	0.6	0.7
96-120	<0.1	<0.1	0.4	0.5
120-144	<0.1	<0.1	0.3	0.4
144-168	<0.1	<0.1	0.3	0.3
0-168	5.4	6.4	36	39
0-168 (standardised)A	6.2	6.4	36	38
Faeces
0-24	76	84	39	35
24-48	3.6	6.3	6.4	6.4
48-72	0.5	0.6	2.1	1.9
72-96	0.4	0.5	1.3	1.3
96-120	0.2	0.2	0.9	1.0
120-144	0.1	0.1	0.7	0.8
144-168	<0.1	0.1	0.6	0.5
0-168	81	92	51	46
Cage wash
0-8	0.3	1.1	1.3	2.8
8-24	0.2	0.2	0.3	0.4
24-48	º<0.1	0.1	0.3	0.3
48-72	º<0.1	º<0.1	0.1	0.2
72-96	º<0.1	º<0.1	0.1	0.2
96-120	º<0.1	º<0.1	0.1	0.1
120-144	º<0.1	º<0.1	º0.1	0.1
144-168	º<0.1	º<0.1	0.1	0.2
0-168	0.6	1.5	2.4	4.1
0-168 (standardised)A	0.7	1.5	2.4	4.0
Total excreted	87	100	90	90
% Absorption (urine po/iv)	18	19	-	-
Carcass	º0.2	º0.2	11	12
Total Recovery	87	100	101	102

° = Mean includes results calculated from data less than 30 dpm above background
A = The 0-168 h urine and cage wash data was standardised to a recovery of 100% for the calculation of
absorption from the urinary data.

Table 2. Mean Blood Concentrations and Pharmacokinetic Parameters Following Single Oral or Intravenous Administration of [14C]-test substance to Rats Expressed as µg Equivalents of test substance/g

Nominal Time after dosing (h)	Group 3 – 1.5 mg/kg oral		Group 4 – 0.5 mg/kg iv
	Male	Female	Male	Female
0.25	0.032	0.037	2.049	2.109
0.5	0.055	0.058	1.728	1.745
1	0.094	0.081	1.625	1.601
2	0.181	0.110	1.496	1.515
4	0.128	0.118	1.523	1.451
8	0.068	0.088	1.288	1.322
12	0.059	0.083	1.141	1.150
24	0.040	0.038	1.152	1.077
48	0.028	0.037	0.992	0.988
72	0.021	0.028	0.831	0.790
Cmax (µg equiv/g)	0.181	0.114	-	-
Cmax/D	0.155	0.0983	-	-
C0	-	-	2.43	2.56
tmax (hours)	2	1.5	-	-
t1/2 (hours)	44.8#	52.7#	107#	98.8#
AUC(0-t) (µg equiv.h/g)	3.15	3.50	78.3	76.4
AUC(0-t)/D	2.71	3.01	173	170
AUC(0-inf) (µg equiv.h/g)	4.50#	5.57#	210#	190#
AUC(0-inf)/D	3.84#	4.80#	462#	422#
AUC % Extrap	30.3#	36.8#	61.2#	59.4#
MRT (hours)	22.0	32.4	32.4	32.2
CL (g/h/kg)	-	-	2.28#	2.39#
CLr (g/h/kg)	-	-	2.04	2.18
Vss (g/kg)	-	-	333#	334#
F (%)	1.57	1.77	-	-

PK parameters are Phoenix (WinNonlin) derived. Parameters generated from individual total radioactivity
concentrations.
# = The extrapolation of the AUC to infinity represents more than 20% of the total area.

 

Conclusions:

Following a single oral administration of [14C]-test substance, the data suggest that differences exist between the fate of an intravenous dose and an oral dose. This is demonstrated in the underestimation of oral bioavailability (ca 2%) from the blood data. This is possibly due to effects of first pass metabolism, enterohepatic recirculation and/or the reactive nature of the test substance in blood and tissues. The fraction of dose systemically available, estimated from the oral:iv urine excretion ratio, was ca 18-19% of the administered dose.

Executive summary:

The excretion and pharmacokinetics of [14C]-test substance was investigated to quantify the absorbed fraction following an oral dose of [14C]-test substance. [14C]-test substance was dosed orally at 1.5 mg/kg and intravenously at 0.5 mg/kg to groups of 4 male and 4 female rats per dose route. Excretion samples were obtained over a 7 day period. After this period, the rats were humanely killed and residual radioactivity was measured in the remaining carcass. Blood samples were taken over a 3 day period to determine the pharmacokinetics of total radioactivity in blood. Male animals had a recovery in the range of 83-88%. Excretion was essentially complete at 168 h post dose with 0.4% or less in the carcass. The low recoveries are likely to be a consequence of the poor homogeneity observed in the dose formulation. This data is accepted and reported throughout. One female animal had a recovery of 87%, the low recovery is likely attributable to a loss of radioactivity from the 8 h urine sample. As this would skew the absorption estimate the animal has been removed from mean calculations. Following a single oral administration of 1.5 mg/kg [14C]-test substance, the majority of administered radioactivity (82-91%) was excreted in the first 24 hours indicating elimination was rapid. The fraction of administered dose systemically available, based on the urinary excretion ratio (oral:iv) accounted for 18-19% of the administered dose. Following a single oral administration of 1.5 mg/kg [14C]-test substance absorption was rapid with peak mean measured blood concentrations observed at 2-4 hours. The oral bioavailability was ca 2% for both sexes suggesting systemic availability was underestimated, as at least 5% of the dose was excreted in urine. No sex related differences were observed in the pharmacokinetics and excretion of total radioactivity. Following a single oral administration of [14C]-test substance, the data suggest that differences exist between the fate of an intravenous dose and an oral dose. This is demonstrated in the underestimation of oral bioavailability (ca 2%) from the blood data. This is possibly due to effects of first pass metabolism, enterohepatic recirculation and/or the reactive nature of the test substance in blood and tissues. The fraction of dose systemically available, estimated from the oral:iv urine excretion ratio, was ca 18-19% of the administered dose.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Sep 1984 to 13 Feb 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Objective of study:

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 - 10 weeks old
- Weight at study initiation: 212 - 241 g
- Housing: During acclimation rats were housed in pairs in stainless steel cages. During the study animals were housed in metabolism cages.
- Diet: available ad libitum. All animals were fasted a minimum of 16 hours before dosing. Animals in each different dose group received food 4 hours after the completion of dosing of each group.
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 60
- Air changes (per hr): ca. 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 Sep 1984 To: 13 Feb 1985

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing suspension was prepared as a stock by dissolving the radiolabelled and nonradiolabelled test substance in solvent, evaporating the solvent, and shaking the solid material. A weighed amount of stock material was suspended in 0.75% methyl cellulose, and made up to a measured volume. Aliquots of the stock solution were taken for concentration and radioactivity determination. Dosing suspension was prepared by mixing the ”hot ” stock dosing suspension with a "cold” stock dosing solution prepared.

Duration and frequency of treatment / exposure:

Single oral dose

Dose / conc.:

5 mg/kg bw (total dose)

Remarks:

2 mL

Dose / conc.:

50 mg/kg bw (total dose)

Remarks:

2 mL

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

2 mL

No. of animals per sex per dose / concentration:

20 females per dose level; 4 females per dose per termination time

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Urine: Urine samples from each set of treated animals were collected at 24, 48, 72, 96, 120, 144 and 168 hours for animals terminated 168 hours after dosing, at 24, 48, 72 and 96 hours for animals terminated 96 hours after dosing, at 24 hours for animals terminated 24 hours after dosing, at 9 hours for animals terminated 9 hours after dosing and at 2 hours for animals terminated 2 hours after dosing. The level of radioactivity in each sample will be determined by liquid scintillation counting.
- Faeces: Faeces from treated animals were collected on the same time schedule as the urine samples. The level of radioactivity was determined after combustion.
- Blood: The blood samples were collected just prior to necropsy by cardiac puncture of animals under halothane anaesthesia. As much blood as possible was taken from each animal and the volume measured. Blood samples were collected only at termination. Heparinized blood was used for radioactivity determinations. It was assumed that the total volume of blood is 64.1 mL per kilogram of body weight
- Tissues: At termination, liver, kidneys, fat, muscle, stomach, small intestine, large intestine, lung, and heart were excised from each animal on the same time schedule as the blood samples. Stomach contents and intestine contents were removed separately from the stomach and large and small intestines with distilled water and stored at -20°C for radioactivity determination later. All the tissues and eviscerated carcasses were quick frozen and stored at – 20°C for radioactive determination later. Each individual metabolism cage was washed with 50% methanol/water and the radioactivity content of the washings determined.

Type:

excretion

Results:

Faeces (main route of excretion): 79 to 84.8% of administered dose, depending on dose level

Type:

excretion

Results:

Urine: 11.5 to 5.4% of the administered dose excreted depending on the dose level

Type:

distribution

Results:

Stomach: retention of 14.4 to 65.5% of administered dose depending on the dose level

Type:

distribution

Results:

Kidneys: 0.07 to 0.71% administered dose depending on the dose level

Details on distribution in tissues:

Measurement of high levels of radioactivity in the stomach indicated that there was a delay in stomach evacuation. The delay was apparent at the mid and high doses but was more pronounced at the high dose. Nine hours after administration of 50 mg/kg, stomach contents contained 25.1% of the administered dose (AD). Stomach evacuation was complete by 24 hours. At a dose of 200 mg/kg, the % AD in stomach contents in female rats at 2 hours was 65.5%, at 9 hours 52.1% and 24 hours 14.4%. The delay in stomach evacuation increased with dose.
With the exception of the gastrointestinal tract tissues, radioactivity was confined mainly to the kidneys. The total percent of the administered dose (AD) found in kidneys was highest at the low dose, but represented only 0.71% AD. Kidney concentrations (as ug equivalents/g) were approximately 4 to 40 times greater than liver concentrations and the level of radioactivity was more
persistent in the kidneys. Kidney or liver concentrations were not proportional to dose when the maximum observed concentrations were examined. At 5 mg/kg, kidney concentrations were greatest at 2 hours post dose (4.94 ug equivalents/g, 0.41% AD/g) and decreased with time. At 50 mg/kg, kidney concentrations were greatest at 9 hours post dose (21.1 ug equivalents/g, 0.17% AD/g) and then decreased with time. At 200 mg/kg, kidney concentrations were greatest at the 24 hour necropsy (34.6 ug equivalents/g, 0.07% AD/g) and then decreased. The rate of depletion of the radioactivity was slower in kidneys than the other tissues.

Details on excretion:

- Faeces: The major route of elimination of the radiolabel was in the faeces at all 3 dose levels. Seventy-nine percent of the 5 mg/kg dose was eliminated in faeces during the 48 hours after dosing and represented 95.6% of the radioactivity in the faeces. At the mid-dose of 50 mg/kg, 84.8% of the dose was eliminated in faeces during the 72 hours after dosing and represented 97.2% of the radioactivity in the faeces. At the high dose of 200 mg/kg, 84.8% of the dose was also eliminated in faeces during the 72 hours after dosing which represented 92.5% of the radioactivity in the faeces. There appeared to be a delay in faecal excretion at both the mid and high doses.
- Urine: The percent of the dose excreted in urine decreased with increasing dose. At the low dose of 5 mg/kg, of approximately 11.5% of the administered dose (%AD) was excreted in urine during the 7 days post dose and urinary excretion was 92% complete (10.56% of the 11.45% AD excreted) in 24 hours. At the mid-dose of 50 mg/kg, approximately 8.8% AD was excreted in urine with excretion 80% complete in 24 hours and 94% complete in 48 hours. At the high dose of 200 mg/kg, only 5.4% of the AD was excreted in urine. The excretion was 57% complete in 24 hours, less than 85% complete in 48 hours and required 72 hours for 95% completion.
There appeared to be a definite relationship between the percent AD excreted in urine and the administered dose because, as the dose increased from 5 to 50 to 200 mg/kg, urine contained 11.5 (0.57 mg/kg ), 8.8 (4.39 mg/kg) and 5.4% (10.86 mg/kg) AD, respectively. The total excretion of radiolabel in the urine was not proportional to dose. At 200 mg/kg the excretion of 10.86 mg/kg was less than half the amount expected (0.57 x 40 = 22.8 mg/kg) based on urinary excretion of the 5 mg/kg dose. The rate of excretion of radiolabel in the urine also appeared to be delayed with respect to increasing dose level. Al though the amount (mg excreted/kg bw) of radiolabel excreted in urine increased from 0 to 24 hours as dose level increased, the increase was not directly proportional to dose. Both the delay in urinary excretion of radiolabel and the decrease in the total percent of the dose excreted as dose level increased suggested that the mechanism of urinary excretion became saturated.

Metabolites identified:

not measured

Animal observations

All animals treated at the 200 mg/kg dose level had loose stools during the 24 hours immediately following dose administration. Loose faeces may have resulted in some contamination of the corresponding urine samples, however, the extent of contamination and its contribution to recovery of radiolabel in urine was not readily determinable. The design of the studies with rats did not allow intermediate observation of large intestinal contents. The presence of soft stools noted with rats, however, suggested that high dose animals may have had watery large intestinal contents. Increased motility or water retention in the lower gastrointestinal tract may have occurred with rats at the high dose of 200 mg/kg.

 

Table 2. Excretion of radioactivity during 168 hours after oral administration of the test substance

% administered dose	Dose level (mg/kg)
	5	50	200
Urine	11.45 ± 1.2	8.78 ± 0.46	5.43 ± 0.55
Feces	82.41 ± 6.72	87.21 ± 4.73	91.65 ± 6.24
Cage wash	0.04 ± 0.01	0.07 ± 004	0.15 ± 0.16
Total	93.90 ± 5.83	96.05 ± 4.31	97.23 ± 6.05

Conclusions:

The major route of elimination of the radiolabel was in the faeces at all 3 dose levels. Seventy-nine percent of the 5 mg/kg dose was eliminated in faeces during the 48 hours after dosing and represented 95.6% of the radioactivity in the faeces. At the mid-dose of 50 mg/kg, 84.8% of the dose was eliminated in faeces during the 72 hours after dosing and represented 97.2% of the radioactivity in the faeces. At the high dose of 200 mg/kg, 84.8% of the dose was also eliminated in faeces during the 72 hours after dosing which represented 92.5% of the radioactivity in the faeces. The percent of the dose excreted with urine at the 5 mg/kg dose level was 11.5%, at the 50 mg/kg dose level 8.8% and at the 200 mg/kg dose level 5.4%.

Executive summary:

To study the absorption, tissue distribution and excretion of radioactivity during the 7 days following a single oral administration of the test substance 60 female Sprague-Dawley CD rats were exposed to several dose levels (5, 50 or 200 mg/kg bw) by oral gavage. Urine and faeces were collected at 24-hour intervals except for animals sacrificed after 2 and 9 hours. All animals were killed by exsanguination under halothane/oxygen anaesthesia. Liver, kidneys, fat, muscle, heart, lungs, stomach, small and large intestines, stomach contents, small intestinal contents and large intestinal contents were removed from each animal and assayed for radioactivity.

Administration of 14C-test substance to female rats at 5, 50 and 200 mg/kg resulted in slow stomach evacuation at the mid and high doses, delayed faecal excretion at the mid and high doses and a decrease in the percent of the administered dose excreted in urine at the mid and high doses compared to the low dose. The major route of elimination of the radiolabel was in the faeces at all 3 dose levels. Seventy-nine percent of the 5 mg/kg dose was eliminated in faeces during the 48 hours after dosing and represented 95.6% of the radioactivity in the faeces. At the mid-dose of 50 mg/kg, 84.8% of the dose was eliminated in faeces during the 72 hours after dosing and represented 97.2% of the radioactivity in the faeces. At the high dose of 200 mg/kg, 84.8% of the dose was also eliminated in faeces during the 72 hours after dosing which represented 92.5% of the radioactivity in the faeces The percent of the dose excreted with urine at the 5 mg/kg dose level was 11.5%, at the 50 mg/kg dose level 8.8% and at the 200 mg/kg dose level 5.4%.

Blood data indicated that there was a delay in time to peak blood concentrations which may have been caused by a delayed or incomplete evacuation of the stomach.

Measurement of high levels of radioactivity in the stomach indicated that there was a delay in stomach evacuation. The delay was apparent at the mid and high doses but was more pronounced at the high dose. With the exception of the gastrointestinal tract tissues, radioactivity was confined mainly to the kidneys.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2011 to 13 Aug 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

excretion

metabolism

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Version / remarks:

1987

Qualifier:

according to guideline

Guideline:

other: 94/79/EC

Version / remarks:

1994

Qualifier:

according to guideline

Guideline:

other: JMAFF 12 Nousan No 8147

Version / remarks:

2000

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Remarks:

Han Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Males: 9 - 12 weeks; females: 9 - 14 weeks
- Weight at study initiation: Males: 282 - 362 g; females: 185 - 211 g
- Housing: Pre-study: multiply housed in solid floored polycarbonate; and stainless steel cages with bedding; During study: multiply housed in polycarbonate and stainless steel cages with raised wire floors. Pre-study (metabolism): singly housed in all-glass metabolism cages suitable for pre-dose sample collection; During study (metabolism): singly housed in all-glass metabolism cages suitable for sample collection.
- Diet: A standard laboratory diet of known formulation was available to all animals ad libitum, with the exception of rats prior to surgery, as they were given rodent chow (14% protein)
- Water: Tap water ad libitum
- Acclimation period: at least 5 days prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1
- Humidity (%): 48 ± 12
- Air changes (per hr): 15
- Photoperiod: Alternating 12-hour light and dark cycles

IN-LIFE DATES: From: 24 May 2011 To: 13 Aug 2012

OTHER: See "Any other information on materials and methods incl. tables" section for description of surgical procedures on test animals prior to dosing.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Prior to the actual dose preparations, a trial [14C]-test substance preparation was formulated at a target dose concentration of 40 mg/mL (the intended concentration for the 200 mg/kg dose groups) in order to assess the suitability of the dose formulation procedures. The trial preparation was also used to assess the stability of the radiochemical in the dose vehicle for up to 21 days post preparation. The trial dose preparation mimicked the procedures required for actual dose preparation but were prepared using the minimum practical quantities of test item. For each dose preparation, an appropriate amount of non-radiolabelled test substance was accurately weighed into a volumetric flask. A radiolabelled stock solution was prepared by dissolving an amount of [14C]-test substance in acetonitrile and removing aliquots to confirm the radioactive concentration. The calculated required volume of stock [14C]-test substance was dispensed into the volumetric flask containing non-radiolabelled test substance and made to volume with acetonitrile. Aliquots were removed to determine the specific activity of the radiodiluted solution. Due to the large volume of acetonitrile required to dissolve test substance in the 40 mg/mL dose formulation, this preparation was transferred to a round bottomed flask with washings of acetonitrile and evaporated down to a volume of ca 50 mL using a rotary evaporator. Each preparation was then transferred to a mortar bowl and the acetonitrile evaporated under a steady stream of nitrogen. The volumetric flask/round bottomed flask was sequentially rinsed with acetonitrile, washings transferred to the mortar bowl and blown down to dryness under a steady stream of nitrogen. As much as possible of the test item was removed from the flask and transferred into the mortar bowl. In each dose preparation, the contents of the mortar bowl was lightly wetted with a small amount of 0.5% (w/v) aqueous CMC (dose vehicle) and ground into a fine paste with a pestle. The paste was transferred to a pre-weighed glass container. Additional dose vehicle was added to the mortar bowl and ground with the pestle before transferring into the glass container. This process was repeated several times before the contents of the glass container was made up to the final dose volume to achieve the required target concentration. Whenever practical, the dose formulation was stirred with a magnetic stirrer until preparation and dose administration were complete.

Duration and frequency of treatment / exposure:

Single oral dose

Dose / conc.:

5 mg/kg bw (total dose)

Remarks:

[14C]-test substance

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

[14C]-test substance

No. of animals per sex per dose / concentration:

5 animals per sex per dose

Control animals:

no

Positive control reference chemical:

None

Details on study design:

- Dose selection rationale: Previous toxicity studies showed that the acute oral median lethal dose of the test substance was in excess of 5000 mg/kg, therefore no adverse effects were expected at 5 or 200 mg/kg.
- Rationale for animal assignment: Group 1 and 2 (5 males and 5 females per group) were used for the pharmacokinetic study; Group (5 males and 5 females per group) 3 and 4 for excretion study; Group 5 and 6 (5 males and 5 females per group) were composed of bile cannulated rats and used for excretion study.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes, bile, expired air, gastrointestinal tract and contents, residual carcasses

Time and frequency of sampling:
- Intact rats:
Urine: predose, 6, 12, 24, 48, 72, 96, 120, 144 and 168 h post dose
Faeces: 24, 48, 72, 96, 120, 144 and 168 h post dose
Cage wash: 24, 48, 72, 96, 120, 144 and 168 h post dose
Expired air: 24 and 48 h post dose
Whole blood and plasma: 168 h post dose
GI tract and contents: 168 h post dose
Carcass: 168 h post dose
- Bile duct cannulated rats:
Urine: predose, 6, 12, 24, 48 and 72 h post dose
Bile: predose, 1, 2, 4, 8, 12, 24, 48 and 72 h post dose
Faeces: predose, 24, 48 and 72 h post dose
Cage wash: 24, 48 and 72 h post dose
GI tract and contents: 72 h post dose
Carcass: 72 h post dose

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, plasma, bile
- Time and frequency of sampling: the same as for pharmacokinetic study
- From how many animals: pooled separately for intact and bile-duct cannulated rats
- Method type(s) for identification: LCMS and TLC
- Limits of detection: 0.01 – 0.5 µg equiv/g

Type:

absorption

Results:

10 - 20 %

Type:

excretion

Results:

75 - 80 % unabsorbed, unchanged test substances excreted in faeces; absorbed dose excreted via biliary system and urine

Type:

metabolism

Results:

hydroxylation (most abundant metabolite 28-37% of the total radioactivity AUC), oxidation of the nitrile groups to amides and glutathione conjugation

Details on absorption:

Following a single 5 mg/kg dose of [14C]-test substance to male and female rats, radioactivity was detectable in blood from the first timepoint (0.5 hours). The Cmax of radioactivity in blood was 0.21 and 0.31 µg equiv/g in male and females, respectively, with tmax at 8 hours post dose in males and 4 hours post dose in females. Concentrations in males declined quickly between 12 and 24 hours, then gradually to 54 h post dose. Concentrations in females maintained a plateau between 8 and 12 hours post dose before declining rapidly at 24 h post dose. Concentrations then decreased steadily to 54 hours post dose, but were still detected. In plasma radioactivity was detectable from the first timepoint (0.5 hours) with the maximum concentration greater than that in blood at 0.52 and 0.58 µg equiv/g in male and females, respectively. The tmax occurred at 8 hours post dose in both males and females after which
concentrations decreased steadily up to 72 hours post dose but was still detected.

Following a single 200 mg/kg dose of [14C]-test substance to male and female rats, radioactivity was detectable in blood from the first timepoint (0.5 hours). The Cmax of radioactivity in blood was 3.2 and 6.0 µg equiv/g in male and females, respectively, with tmax at 12 hours post dose in both males and females. Concentrations in males declined steadily until 54 hours post dose and had fallen below the limit of detection by 72 hours post dose. Concentrations in females also declined steadily and had fallen below the limit of detection by 48 hours post dose. In plasma radioactivity was detectable from the first timepoint (0.5 hours). The Cmax was greater than that in blood at 6.8 and 11.6 µg equiv/g in male and females, respectively. The tmax was observed at 12 hours post dose in males and 8 hours post dose in females, after which concentrations in both males and females decreased steadily up to 96 hours post dose.

Details on excretion:

See "Any other information on results incl. tables" section

Test no.:

#1

Toxicokinetic parameters:

Cmax: blood: 0.21 and 0.31 µg equiv/g in male and females

Remarks:

5 mg/kg bw dose

Test no.:

#1

Toxicokinetic parameters:

Tmax: blood: 8 hours post dose in males and 4 hours post dose in females

Remarks:

5 mg/kg bw dose

Test no.:

#1

Toxicokinetic parameters:

Cmax: plasma: 0.52 and 0.58 µg equiv/g in male and females, respectively

Remarks:

5 mg/kg bw dose

Test no.:

#1

Toxicokinetic parameters:

Tmax: plasma: 8 hours post dose in both males and females

Remarks:

5 mg/kg bw dose

Test no.:

#2

Toxicokinetic parameters:

Cmax: blood: 3.2 and 6.0 µg equiv/g in male and females, respectively

Remarks:

200 mg/kg bw dose

Test no.:

#2

Toxicokinetic parameters:

Tmax: blood: 12 hours post dose in both males and females

Remarks:

200 mg/kg bw dose

Test no.:

#2

Toxicokinetic parameters:

Cmax: plasma: 6.8 and 11.6 µg equiv/g in male and females, respectively

Remarks:

200 mg/kg bw dose

Test no.:

#2

Toxicokinetic parameters:

Tmax: plasma: 12 hours post dose in males and 8 hours post dose in females

Remarks:

200 mg/kg bw dose

Metabolites identified:

yes

Details on metabolites:

The metabolism of the test substance was extensive with 7 metabolites formed through oxidation, hydroxylation and conjugation identified. For each matrix, metabolites were qualitatively and quantitatively similar. The metabolites identified were products of oxidation of the nitrile to an amide, two acetyl-cysteine conjugates, hydroxylated test substance (most abundant, accounting for 28-37% of the total radioactivity AUC), amide with a sulphonic acid and amide with 2 sulphonic acid groups. Metabolite profiles were qualitatively and quantitatively similar regardless of sex and dose rate.

Excretion of radioactivity from intact rats

5 mg/kg

Following a single oral administration of 5 mg [14C]-test substance/kg, the major route of elimination was via the faeces, with 85-91% of the administered radioactivity recovered by 168 hours post dose. Urinary excretion accounted for 5.5-7.0% of the administered dose. Recovery in cage washings were minimal accounting for <1.6% of the dose. Total radioactivity excreted via expired air was negligible with <0.1% of the dose recovered after 48 hours post dose. The majority (ca 93%) of the administered radioactivity was excreted by 96 h post dose. Excretion was essentially complete by 168 h post dose with <0.2% remaining in the carcass and <0.1% remaining in the gastrointestinal tract. The total recovery of administered radioactivity including excreta, cage wash, gastrointestinal tract and residual carcass was 99 and 92% in males and females, respectively.

 

200 mg/kg

Following a single oral administration of 200 mg [14C]-test substance/kg, the major route of elimination was via the faeces, with 99-115% of the administered radioactivity recovered by 168 hours post dose. Urinary excretion accounted for <3.0 % of the administered dose. Recovery of radioactivity in cage washings were minimal accounting for <1.0% of the dose. Radioactivity excreted via expired air was negligible with <0.1% recovered after 48 hours post dose. The majority (>91%) of the administered radioactivity was excreted by 48 h post dose with excretion essentially complete by 168 h post dose with <0.2% remaining in the carcass and the gastrointestinal tract. The total recovery of administered radioactivity including excreta, cage wash, gastrointestinal tract and residual carcass was 118 and 104% in males and females, respectively.

 

Excretion of Radioactivity from Bile Duct Cannulated Animals

5 mg/kg

Following a single oral administration of 5 mg [14C]-test substance/kg to bile duct cannulated rats, the major route of elimination was via the faeces, with 75-80% of the administered radioactivity recovered by 72 hours post dose. Biliary elimination accounted for 12% of the administered dose and urinary excretion accounted for 5.8-10% of the administered dose. Recovery in cage washings were minimal accounting for <1.6% of the dose. The majority (>94%) of the administered radioactivity was excreted by 48 h post dose with excretion essentially complete by 72 h post dose. Less than 0.5% and 1% remained in the carcass and gastrointestinal tract, respectively. The total mean recovery of administered radioactivity including excreta, cage wash, gastrointestinal tract and residual carcass was 99% in both males and females.

 

200 mg/kg

Following a single oral administration of 200 mg [14C]-test substance/kg to bile duct cannulated rats, the major route of elimination was via the faeces, with 81-95% of the administered radioactivity recovered by 72 hours post dose. Biliary elimination accounted for 4.9-7.5% and urinary excretion accounted for 2.9-4.3% of the administered dose. Recovery in cage washings were minimal accounting for <1.6% of the dose. The majority (>93%) of the administered radioactivity was excreted by 48 h post dose with excretion essentially complete by 72 h post dose. Less than 0.3% and 0.2% remained in the carcass and gastrointestinal tract, respectively. The total mean recovery of administered radioactivity including excreta, cage wash, gastrointestinal tract and residual carcass was 104 and 95% in males and females, respectively. 

Metabolism

Table 1. Metabolites of the test substance identified in the study.

Name	Identity
M4	hydroxylated test subtance
M5	dehalogenated test substance amide
M6	acetyl-cysteine conjugate
M7	acetyl-cysteine conjugate
M8	dehalogenated + amide
M9	amide + sulphonic acid
M10	amide + 2sulphonic groups
M11	unidentified
M12	unidentified

Conclusions:

Following oral administration of [14C]-test substance to male and female rats, the majority of administered dose was unabsorbed (75-80% of the 5 mg/kg dose) and excreted as unchanged test substance in the faeces. Absorption rate varied between 10 % for 200 mg/kg bw dose and 20 % for 5 mg/kg bw dose. Metabolite profiles were qualitatively and quantitatively similar regardless of sex and dose. The test substance was metabolized via hydroxylation, oxidation of the nitrile groups to amides and glutathione conjugation. In plasma, the hydroxylated test substance was the most abundant metabolite accounting for 28-37% of the total radioactivity AUC. The products of oxidation of the nitrile groups to amides as well as glutathione conjugates were each accounting for 5.5-17% of the TRA. The absorbed dose was excreted via biliary elimination and in urine, with no metabolite individually accounting for >6% of the total excreted dose and all unidentified metabolites and origin bound material each at less than 5% of the dose.

Executive summary:

The pharmacokinetics, absorption, metabolism and excretion of total radioactivity were investigated following a single oral administration of [14C]-test substance (5 or 200 mg/kg) to male and female Han Wistar rats. To characterise the pharmacokinetics of total radioactivity, blood and plasma were collected at intervals up to 3 days post dose. To characterise the absorption and excretion of total radioactivity, urine and faeces were collected at intervals up to 7 days post dose and in bile up to 3 days post dose. The nature and identity of metabolites present in samples of plasma, urine, bile and faeces was investigated. Following a single 5 mg/kg dose of [14C]-test substance to male and female rats, radioactivity was detectable in blood from the first timepoint (0.5 hours). The Cmax of radioactivity in blood was 0.21 and 0.31 µg equiv/g in male and females, respectively, with tmax at 8 hours post dose in males and 4 hours post dose in females. Concentrations in males declined rapidly between 12 and 24 hours, then gradually to 54 h post dose. Concentrations in females maintained a plateau between 8 and 12 hours post dose before declining rapidly at 24 h post dose. Concentrations then decreased steadily to 54 hours post dose. Radioactivity was still detected in blood in both male and female rats at 54 hours post dose. Concentrations of radioactivity were detectable in plasma from the first timepoint (0.5 hours). The Cmax of radioactivity in plasma was greater than that in blood at 0.52 and 0.58 µg equiv/g in male and females, respectively. The tmax occurred at 8 hours post dose in both males and females after which concentrations decreased steadily up to 72 hours post dose. Radioactivity was still detected in plasma in both male and female rats at 72 hours post dose.
Following a single 200 mg/kg dose of [14C]-test substance to male and female rats,radioactivity was detectable in blood from the first timepoint (0.5 hours). The Cmax of radioactivity in blood was 3.2 and 6.0 µg equiv/g in male and females, respectively, with tmax at 12 hours post dose in both males and females. Concentrations in males declined steadily until 54 hours post dose and had fallen below the limit of detection by 72 hours post dose.Concentrations in females also declined steadily, however had fallen below the limit ofdetection by 48 hours post dose. Radioactivity was detectable in plasma from the first time point (0.5 hours). The Cmax of radioactivity in plasma was greater than that in blood at 6.8 and 11.6 µg equiv/g in male and females, respectively. The tmax occurred at 12 hours postdose in males and 8 hours post dose in females, after which concentrations in both males and females decreased steadily up to 96 hours post dose. Following a single oral administration of 5 mg [14C]-test substance/kg, the major route of elimination was via the faeces, with 85-91% of the administered radioactivity recovered by168 hours post dose. Urinary excretion accounted for 5.5-7.0% of the administered dose.
Recovery in cage washings were minimal, accounting for <1.6% of the dose. Total metabolites and solvent system 2 (SS2) was used separate polar metabolites that were origin bound in SS1. The most abundant metabolites were confirmed by LCMS. The test substance was metabolised in the rat giving up to 7 metabolites through oxidation, hydroxylation and conjugation. For each matrix, metabolites were qualitatively and quantitatively similar. The majority of the dose was excreted as unabsorbed parent in faeces. The major metabolites were identified as M5 (dehalogenated test substance amide) and M6 (acetyl-cysteine conjugate). Other identified components included M4 (hydroxylated test substance), M7 (acetyl-cysteine conjugate), M8
(dehalogenated + amide), M9 (amide + sulphonic acid) and M10 (amide + 2 sulphonic groups). M11 and M12 had similar Rf (ca0.2) and these could not be identified or quantified as individual metabolites. Metabolite profiles were qualitatively and quantitatively similar regardless of sex and dose.
The most abundant component excreted in urine was M6 (≤ 3.3% dose). M5 and the region comprising of M7 and/or M10 accounted for ≤ 1.5% dose. A single component remained unidentified (≤ 1.0% dose) and origin bound material accounted for ≤ 1.2% dose. In bile, M6 accounted forca≤ 1.7% dose and M5 and M7/M10 accounted for (≤ 1.4% dose). Three components in bile remained unidentified (≤ 2.0% dose) and origin bound material accounted for ≤ 2.6% dose. The most abundant component excreted in faeces was unabsorbed test substance (ca 5.2-64.3% dose). M8, M4, M5, M6 and M7/M10 were minor metabolites (≤ 5.4% dose). Two components remained unidentified (≤ 1.6% dose) and origin bound material accounted for ≤ 4.4% dose. The most abundant component circulating in plasma was M4 which accounted for 29-38% of the total radioactivity AUC (TRA). The other circulating components observed were test substance at 5.5-11% TRA, M8 at 11-17% TRA and M9 at 4.7-12% TRA. Two components remained unidentified (4.1-13% TRA, ≤ 0.082 µg/g) and origin bound material accounted for 7.8-16% TRA, ≤ 0.181 µg/g. Following oral administration of [14C]-test substance to male and female rats, the majority of administered dose was unabsorbed (75-80% of the 5 mg/kg dose) and excreted as unchanged test substance in the faeces. Metabolite profiles were qualitatively and quantitatively similar regardless of sex and dose. The test substance was metabolised via hydroxylation (M4), oxidation of the nitrile groups to amides (M8 and M5) and glutathione conjugation (M6, M7, M9 and M10). In plasma, M4 was the most abundant metabolite accounting for 28-37% of the total radioactivity AUC, with M8, test substance and M9 each accounting for 5.5-17% of the TRA. The absorbed dose was excreted via biliary elimiation and in urine, with no metabolite individually accounting for >6% of the total excreted dose and all unidentified metabolites and origin bound material each at less than 5% of the dose.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Nov 1983 to 30 Jan 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Objective of study:

absorption

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 252-271 g
- Housing: During acclimation rats were housed in pairs in stainless steel cages. During the study animals were maintained in Bollman restraint cages.
- Diet: ad libitum; all animals were fasted 16 h prior to operation, after the operation a 4% dextrose/0.18% NaCl solution was substituted for solid food and provided ad libitum until termination
- Water: ad libitum; 4% dextrose/0.18% NaCl solution was substituted for water and provided ad libitum until termination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 - 60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A microparticulate suspension with a particle size of 3.8 microns was prepared by mixing 2 individual vials which had essentially the same concentration (1.005 ± 0.045 mg/mL) and specific activity (28.65 ± 0.45 µCi/mg). The final dosing suspension contained 1.01 mg/mL with a specific activity of 27.9 µCi/mg. The specific activity was within 3% of the theoretical value of 28.65 µCi/mg.

Duration and frequency of treatment / exposure:

Single oral dose

Dose / conc.:

5 mg/kg bw (total dose)

Remarks:

1 mL per animal

No. of animals per sex per dose / concentration:

See "Any other information on materials and methods incl tables"

Control animals:

no

Positive control reference chemical:

None

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, bile and carcass
- Time and frequency of sampling: bile - from administration until 48 h post-administration in 60 minute intervals; blood - 6 and 24 h post dose, as much blood as possible was taken upon termination; urine and faeces samples - 6, 24 and 48 h after dosing
- Method type for identification: Liquid scintillation counting
- Limits of detection and quantification:
- Other: At termination the gastrointestinal tract was separated from each carcass

Type:

absorption

Results:

Dose absorbed was calculated using the data from the bile, urine, carcasses and cage washings. When these values were combined and corrected for overall recovery (91.24%) of radioactivity, the data indicated that 33.95 ± 4.95 % of the dose was absorbed.

Type:

excretion

Results:

21% of the dose in bile and 7.6% of the dose in urine

Details on absorption:

For male rats 31.67 ± 5.37% of a 5 mg/kg dose was absorbed and there was essentially no difference between infused and non-infused animals. Correction for recovery indicated that as much as 34.72 ± 5.88% of the dose was absorbed. For female rats 29.58 ± 1.91% of the dose was absorbed. When the value was corrected for recovery, then the absorbed amount was 32.42 ± 2.10% of the dose for female rats. Differences in the values for male and female rats probably represented animal variation. It has been concluded that Sprague-Dawley rats absorb 33.95 ± 4.95% of a 5 mg/kg dose of the test substance.

Details on excretion:

BILIARY EXCRETION
Male Sprague-Dawley rats excreted 21.1% of an orally administered dose of 14C-test substance in the bile during the 48 hours after dosing. The infusion of sodium taurocholate did not appear to have an effect on the percent of the administered dose (% AD) that was excreted in bile. The bile contained 20.04 ± 2.68% of the administered radioactivity (at a dose of 5 mg/kg) after 48 hours in the absence of taurocholate. For animals infused with taurocholate 22.08 ± 3.64% of the administered radioactivity was found in the bile. These values were considered to be indistinguishable and the biliary excretion of radioactivity was 21.06 ± 3.15% AD for the 8 male rats. Approximately 56% of the total excreted radioactivity was excreted within 4 hours (11.88/21.06) and excretion was greater than 93% complete (19.69/21.06) within 24 hours. Although taurocholate did not appear to have an effect on the total amount of radiolabel excreted in bile, it did have an effect on the amount of bile excreted by the male rats, rats infused with taurocholate excreted almost twice the volume (53.72 ± 7.78 mL) of bile in 48 hours as male rats that did not receive taurocholate (28.21 + 5.02 mL). Therefore, the radioactivity was more concentrated in the bile of noninfused rats. Measurable biliary levels were obtained 1 hour after dosing and maximum concentrations of radiolabel occurred within approx. 2 h. The concentration of radioactivity (as ug eq/mL) decreased rapidly to less than 10% of maximum by 12 hours post-dose. For female Sprague-Dawley rats, the effects of taurocholate infusion on biliary excretion of radioactivity were inconclusive. The 2 rats infused with taurocholate appeared to excrete more radiolabel in bile (17.94 ± 1.01% AD) than the 2 rats which did not receive taurocholate (15.41 0.59% AD). However, this apparent difference in biliary excretion may have resulted from the small number of animals used rather than from the presence or absence of taurocholate. The overall excretion for the two groups was 16.67 ± 1.61% AD. Biliary excretion of radiolabel was approximately 49% complete within 4 hours and was greater than 96% complete within 24 hours. As had been noted with male rats, taurocholate also had an effect on the amount of bile excreted by female rats. In the presence of taurocholate, 47.4 mL of bile were excreted in 48 hours, whereas, in the absence of taurocholate, 29.6 mL of bile were excreted in 48 hours. The maximum concentration in bile occurred at 1 hour post dose for female rats infused with taurocholate. The concentration decreased to about 12% of maximum by 12 hours. The maximum concentration at 1 hour for female rats infused with taurocholate (50.25 ug eq/mL) was similar to the maximum concentration at 2 hours for male rats infused with taurocholate (51.97 ug eq/mL). The bile of female rats that did not receive taurocholate did not appear to be as concentrated as the bile of female rats that did receive taurocholate. The highest concentration, 32.44 ug eq/mL, occurred at 2 hours and was significantly. Less than the maximum concentration, 50.25 ug eq/mL, for female rats infused with taurocholate. However, higher concentrations of radiolabel in bile were more persistent in the non-infused females, and by 12 hours the concentrations of radiolabel in bile had decreased to only 23% of the maximum concentration.

URINARY EXCRETION
The urine of the male rats contained 7.64 ± 0.84% AD after 48 hours. Infusion of sodium taurocholate had no effect on urinary excretion. The urinary data from one non-infused animal were not used to calculate the mean urinary excretion for male rats, because the levels of radioactivity were inordinately high compared to all other male rats (60% greater than the mean % AD excreted by the other 7 males). The urine of female rats contained 11.71 ± 0.82% AD after 48 hours. Infusion of sodium taurocholate had no effect on urinary excretion. The urine of one of the non-infused animals contained much less radioactivity after 6 hours than the other rats, but the levels of radioactivity were similar for all female rats after 24 and 48 hours.

RADIOACTIVITY IN FAECES
The faeces represented the major route of elimination of the radio-labelled dose of the test substance for male and female rats during this 48-hour study and contained approximately 54% AD. When the gastrointestinal tract was taken into consideration, about 60% of the radioactivity was eliminated via the faeces.

Metabolites identified:

not measured

Table 1. Recovery of radioactivity (as % administered dose) from rats administered 5 mg/kg bw test substance

Rat group	% in bile	% in urine(a)	% in carcass	% absorbed	% in faeces	% in GI tract	% non-absorbed	Total %
Males	20.04 ± 2.68	7.71(b) ± 0.79	3.73 ± 3.73	31.67 ± 6.14	47.49 ±17.34	10.22 ±9.83	57.71 ±7.56	89.18 ±4.9
Males + SC	22.08 ±3.64	7.92 ±1.02	1.88 ±1.25	31.87 ±5.43	53.09 ±9.9	6.07 ±5.46	59.15 ±6.53	91.03 ±3.62
All males	21.06 ±3.15	7.83 (b) ±0.86	2.8 ±2.76	31.67 ±5.37	50.29 ±13.41	8.14 ±7.69	58.43 ±6.58	90.1 ±4.11
Females	15.41 ±0.59	11.63 ±0.21	1.03 ±0.55	28.07 ±1.35	61.62 ±8.73	2.4 ±0.64	64.02 ±8.1	92.08 ±6.75
Females + SC	17.94 ±1.01	12.27 ±1.10	0.89 ±0.16	31.09 ±0.08	60.59 ±2.46	3.27 ±0.16	63.86 ±2.3	94.94 ±2.22
All females	16.67 ±1.61	11.95 ±0.74	0.96 ±0.34	29.58 ±1.91	61.10 ±5.27	2.83 ±0.63	63.94 ±4.86	93.51 ±4.42
Mean % absorbed	30.97± 4.52     33.95± 4.95		Mean % non- absorbed	60.26± 6.43     66.06± 7.06		Mean recovery	91.24± 4.35
Corrected for recovery			Corrected for recovery

a – urine plus cage washes

b - Value does not include data from an animal with urinary excretion 60% greater than the mean of the other male rats.

SC – supplement with sodium taurocholate

Conclusions:

At a dose level of 5 mg of the test substance per kilogram of body weight, male rats excreted approximately 21% of the dose in bile and 7.6% of the dose in urine. Biliary excretion apparently was independent of the presence or absence of bile salts. For female rats, biliary excretion may have been dependent upon sodium taurocholate infusion, but the data were inconclusive. Female rats infused with taurocholate excreted 17.9% of the dose in bile whereas non -infused female rats excreted 15.4% of the dose in bile. The mean biliary excretion for female rats was 16.7% of the dose, Urinary excretion was essentially the same for all female rats, 11.7% AD. The radioactivity was excreted rapidly in bile by both sexes. Peak concentrations of radiolabel were found in bile within approximately 2 hours post-dose for all animals. The only effect of taurocholate infusion appeared to be the volume of bile that was excreted. The amount of the 5 mg/kg dose that was absorbed by the animals was calculated using the data from the bile, urine, carcasses and cage washings. When these values were combined and corrected for overall recovery (91.24%) of radioactivity, the data indicated that 33.95 ± 4.95 % of the dose was absorbed. The remainder of the radioactivity which was found in the faeces and gastrointestinal tract was 66.06 ± 7.06% of the dose and was non-absorbed material.

Executive summary:

Sprague-Dawley rats (252-271 grams) were administered the 14C-test substance by oral gavage at a dose level of 5 mg/kg. The test substance was administered in 0.75% methylcellulose (400 c.p.) as a microparticulate suspension with a mean particle size of 3.8 microns. Bile was collected continuously in fractions of 60 minute duration for 48 hours after dosing. The bile was collected from 8 male rats of which 4 had sodium taurocholate infused and from 4 female rats of which 2 had sodium taurocholate infused. Blood and urine samples were collected at 6, 24 and 48 hours post-dose and faeces were collected at 24 and 48 hours. The gastrointestinal tract was separated from each carcass at termination. Levels of radioactivity were determined in each bile, blood, urine and faecal sample and in the gastrointestinal tracts, carcasses and cage washes. Male rats excreted 21.1% of the dose in bile and about 7.6% in the urine. Biliary excretion appeared to be independent of taurocholate infusion. Female rats excreted 16.7% of the dose in bile and 11.7% of the dose in urine. Biliary excretion by female rats may have had a slight dependence upon the presence of bile salts. The excretion of radiolabel in bile was rapid with peak concentrations in bile within 2 hours after dosing for both sexes. Mean blood concentrations at 6, 24 and 48 hours were similar in all animals but were highly variable at each sampling time. The blood samples taken at 6 hours had the highest concentration of radioactivity and decreased with time. The maximum blood concentration represented less than 0.4 percent of the administered radiolabel. Approximately 91.2% of the administered radioactivity was recovered from the animals on study. The amount of the 5 mg/kg dose that was absorbed by the animals was calculated and corrected for overall recovery of radioactivity. The data indicated that 33.95 ± 4.95% of the dose was absorbed. The remainder of the radioactivity (66.06 ± 7.06)% was found in the faeces and gastrointestinal tract and was non-absorbed material. 

Reference 5

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2013 to 28 Jun 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

2004

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 28. The Conduct of Skin Absorption Studies

Version / remarks:

2004

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Note No. 156. Dermal Absorption.

Version / remarks:

2011

Qualifier:

according to guideline

Guideline:

other: EFSA Guidance on Dermal Absorption.

Version / remarks:

2012

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Vehicle:

water

Duration of exposure:

24 hours

Doses:

- Nominal doses: concentrate formulation: 400 g/L, three different spray dilutions: 10 g/L, 1.875 g/L and 0.667 g/L (equivalent to 1/40, 1/213.3 or 1/600 w/v)
respectively.
- Dose volume: 25.4 µL
- Rationale for dose selection: The application rates and exposure conditions used in this study were designed to simulate predicted normal human exposure to the test material

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: See "Any other information on materials and methods incl tables" section

APPLICATION OF DOSE: The test substance formulations were applied to the skin surface by volume (target =10 μL/cm2) using a suitable positive displacement pipette. The weight of the dose applied was recorded and used for calculations. The concentrations were expressed as g/L using the specific gravity of 1.22 for the formulation concentrate and 1.00 for the dilutions.

TEST SITE
- Area of exposure: 2.54 cm2
- Time intervals for sampling: Samples (0.5 mL) of receptor fluid were taken from the receptor chambers of the static cell system at pre-treatment and at 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 hours after application using an autosampler. The receptor fluid in the chambers was stirred continuously and the receptor volume was maintained by the replacement of a volume of fresh receptor fluid, equal to the sample volume, after each sample had been taken.

ANALYSIS
- Method type for identification: Liquid scintillation counting

OTHER:
- Measurement of mass balance: After the final receptor fluid sample had been taken at 24 hours, the remaining fluid in the receptor chamber was discarded. To remove residual receptor fluid from under the surface of the skin, the receptor chamber was again refilled with fresh receptor fluid (approximately 5 mL), which was afterwards discarded.
The donor chamber was carefully removed and the underside (surface contact with the membrane) wiped with at least a single sponge pre-wetted with 3% Teepol L® in water which was added to the wash sponges (below). The donor chambers were washed with acetone and the sample of the washing taken for analysis by LSC.
The epidermal surface of the skin was decontaminated by gently swabbing the application site with natural sponges pre-wetted with 3% Teepol L® in water, and with further sponges pre-wetted with water. This continued until the decontamination appeared complete or until it was apparent that radiolabel may be being extracted from the epidermis (this was following assessment of radioactivity levels on the skin surface and/or on the sponges with a Geiger counter during the procedure). The sponges were digested in Soluene 350® and a sample taken for analysis by LSC. The surface of the skin was allowed to dry naturally.
- To assess penetration through human stratum corneum, successive layers of the stratum corneum were removed by the repeated application of adhesive tape to a maximum of five strips. The total number of tape strips was recorded. The 5 strips were extracted individually for approximately 20 hours in acetone. The extracts were sequentially numbered and analysed by LSC.
- The remaining skin was carefully removed from the receptor chamber and were digested separately in Soluene 350®. The digest was made up to a recorded volume and a sample taken for analysis by LSC

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: human
- Preparative technique: Human skin samples were obtained from a tissue bank. Skin membranes were cut from the samples at a thickness setting of 400 µm using an electric dermatome.
- Thickness of skin (in mm): 0.4
- Membrane integrity check: Skin integrity was determined by measurement of the electrical resistance across the skin membrane. Membranes with a measured resistance of <10 kΩ (Davies et al, 2004) were regarded as having a lower integrity than normal and not used for exposure to the test materials, due to the possibility of compromised barrier function.
- Storage conditions: -20ºC, on aluminium foil until required for use

PRINCIPLES OF ASSAY
- Diffusion cell: static glass diffusion cell with an exposed membrane area of 2.54 cm2 and a receptor fluid volume of approximately 4.5 mL
- Receptor fluid: 50% ethanol in water
- Solubility od test substance in receptor fluid: 0.02376 mg/mL of the test substance was found to be fully soluble in 50% ethanol water, which is equivalent to 20433% of the total amount of formulation concentrate actually absorbed per cell in a nominal 4.5mL of receptor fluid being absorbed.
- Test temperature: 32ºC ± 1ºC
- Other: discs of approximately 3.3 cm diameter of prepared skin samples were mounted, dermal side down, in such diffusion cells held together with individually numbered clamps and placed in a water bath

Absorption in different matrices:

400 g test substance/L formulation concentrate
The mean absorption rate of the test substance from the formulation concentrate through human dermatomed skin was 0.008 µg/cm²/h over 24 hours. The amounts of the test substance absorbed at 6, 8, 12 and 24 hours were 0.049, 0.072, 0.096 and 0.206 µg/cm2, respectively. These respective amounts, expressed as percentages of the applied dose, equated to 0.001, 0.002, 0.003 and 0.006%.

10 g test substance/L Aqueous spray dilution 1 (1/40 w/v)
The mean absorption rate of the test substance from the 10 g/L aqueous dilution through human dermatomed skin was 0.005 µg/cm²/h during the 24 hour exposure period. The amounts of test substance absorbed at 6, 8, 12 and 24 hours were 0.025, 0.038, 0.058 and 0.120 µg/cm2, respectively. These respective amounts, expressed as percentages of the applied dose, equated to 0.025, 0.037, 0.057 and 0.119%.

1.875 g test substance/L Aqueous spray dilution 2 (1/213.3 w/v)
The mean absorption rate of the test substance from the 1.875 g/L aqueous dilution through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour exposure period. The amounts of test substance absorbed at 6, 8, 12 and 24 hours were 0.019, 0.026, 0.034 and 0.056 µg/cm2, respectively. These respective amounts, expressed as percentages of the applied dose, equated to 0.100, 0.131, 0.174 and 0.285%.

0.667 g test substance/L Aqueous spray dilution 3 (1/600 w/v)
The mean absorption rate of Test substance from the 0.667 g/L aqueous dilution through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour exposure period. The amounts of test substance absorbed at 6, 8, 12 and 24 hours were 0.013, 0.018, 0.027 and 0.054 µg/cm2, respectively. These respective amounts, expressed as percentages of the applied dose, equated to 0.189, 0.261, 0.390 and 0.765%.

Total recovery:

400 g test substance/L formulation concentrate
A mean of 105% of the applied test substance was washed off the skin after 6 hours, with a further 0.238% removed at 24 hours. Small proportions were recovered from the remaining study compartments. A mean of 0.039% of the dose applied was recovered from the donor chamber, tape strips 1-2 (0.009%), tape strips 3-5 (0.007%) and 0.401% was found in the remaining skin. The mean total recovery was 105% of the dose applied.

10 g test substance/L Aqueous spray dilution 1 (1/40 w/v)
A mean of 90.6% of the applied test substance was washed off the skin after 6 hours, with a further 4.39% removed at 24 hours. Small proportions were recovered from the remaining study compartments. A mean of 0.299% of the dose applied was recovered from the donor chamber, tape strips 1-2 (0.181%), tape strips 3-5 (0.165%) and 7.89% was found in the remaining skin. The mean total recovery was 104% of the dose applied.

1.875 g test substance/L Aqueous spray dilution 2 (1/213.3 w/v)
A mean of 99.7% of the applied test substance was washed off the skin after 6 hours, with a further 1.61% removed at 24 hours. Small proportions were recovered from the remaining study compartments. A mean of 0.076% of the dose applied was recovered from the donor chamber, tape strips 1-2 (0.119%), tape strips 3-5 (0.182%) and 6.31% was found in the remaining skin. The mean total recovery was 108% of the dose applied.

0.667 g test substance/L Aqueous spray dilution 3 (1/600 w/v)
A mean of 85.1% of the applied test substance was washed off the skin after 6 hours, with a further 4.69% removed at 24 hours. Small proportions were recovered from the remaining study compartments. A mean of 0.322% of the dose applied was recovered from the donor chamber, tape strips 1-2 (0.145%), tape strips 3-5 (0.412%) and 10.8% was found in the remaining skin. The mean total recovery was 102% of the dose applied

Key result

Time point:

24 h

Dose:

0.667 g/L

Parameter:

rate

Absorption:

12.12 %

Remarks on result:

other: This value is derived from: reception fluid + exposed skin + skin strips

Table 1. Distribution of the test substance in the Test System (% administered dose)

Formula	400 g/L		10 g/L		1.875 g/L		0.667 g/L
Samples (test compartment)	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
Sum strips 1-2	0.009	0.207	0.181	3.7	0.119	5.17	0.145	4.46
Sum strips 3-5	0.007		0.165		0.182		0.412
Remaining skin	0.401		7.89		6.31		10.8
Receptor fluid	0.006		0.119		0.293		0.765
sum absorption	0.423		8.355		6.904		12.122

Conclusions:

The results obtained in this study demonstrate that the absorption of the test substance through human dermatomed skin following the application of the test substance/Azoxystrobin is slow and the vast majority of the test substance can be washed off the skin by normal decontamination procedures. These data predict that the dermal absorption of the test substance from potential exposure to this formulation concentrate and its aqueous spray strength dilutions would be generally low and will not exceed 12.12%.

Executive summary:

The absorption and distribution of test substance from a test substance/Azoxystrobin suspension concentrate (SC) formulation was measured in vitro through human dermatomed skin conforming to the OECD 428 testing guideline. The doses were applied to the dermatomed skin as the test substance/Azoxystrobin formulation concentrate containing 400 g test substance/L and three aqueous spray strength dilutions: nominally containing 10 g, 1.875 g and 0.667 g test substance/L: equivalent to 1/40, 1/213.3 and 1/600 w/v, respectively. The formulation and the aqueous spray strength dilutions were applied at rates of 10 µL/cm2 and left unoccluded for an
exposure period of six hours and a total run time of 24 hours. The absorption process was followed by taking samples of the receptor fluid (50% ethanol in water) at recorded intervals throughout the exposure period. At the end of the experiment, the distribution of test substance in the test system was assessed, which included a tape stripping technique to determine its distribution in the stratum corneum and in the remaining skin.
The formulation concentrate was included to assess exposure to mixer/loaders. The spray strength dilutions used (10 g, 1.875 g and 0.667 g test substance/L) represented typical in use concentrations. These applications were designed to simulate potential human dermal exposure to the formulation during normal use.
The absorption and distribution were followed using [14C]-test substance which was incorporated into the doses prior to application. The samples were analysed by liquid scintillation counting (LSC).
The surface of the dermatomed skin was decontaminated after a six hour exposure period to investigate the amount of test substance absorbed by the end of a typical ‘working day’ period. After decontamination, the absorption of test substance was monitored for the remainder of the 24 hour observation period.

Absorption of test substance from the formulation suspension concentrate (400 g test substance/L) through human dermatomed skin was at a mean absorption rate of 0.008 µg/cm²/h during the 24 hour experimental period. The amount absorbed into the receptor fluid at 24 hours following a six hour wash was 0.206 µg/cm² (0.006% of the dose applied). The vast majority of the applied test substance (mean 105%) was washed off the skin at six hours, and an additional 0.238% was removed in the skin wash after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.039%), tape strips 1-2 (0.009%), tape strips 3-5 (0.007%) and 0.401% was found in the remaining skin. The mean total recovery was 105% of the dose applied.
The absorption rate of test substance from the 10 g/L aqueous dilution 1 through human dermatomed skin was 0.005µg/cm²/h during the 24 hour experimental period. The amount absorbed into the receptor fluid at 24 hours, following a six hour wash, was 0.120µg/cm² (0.119% of the dose applied). The majority of the applied test substance (mean 90.6%) was washed off the skin at six hours, and an additional 4.39% was removed in the skin wash after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.299%), tape strips
1-2 (0.181%), tape strips 3-5 (0.165%) and 7.89% was found in the remaining skin. The mean total recovery was 104% of the dose applied.

The absorption rate of test substance from the 1.875 g/L aqueous dilution 2 through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour experimental period. The amount
absorbed into the receptor fluid at 24 hours following a six hour wash was 0.056µg/cm² (0.285% of the dose applied). The proportion of the applied test substance that was washed off the skin at 6 hours was 99.7% with an additional 1.61% being removed by skin washing after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.076%), tape strips
1-2 (0.119%), tape strips 3-5 (0.182%) and 6.31% was found in the remaining skin. The mean total recovery was 108% of the dose applied.

The absorption rate of test substance from the 0.667 g/L aqueous dilution 3 through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour experimental period. The amount absorbed into the receptor fluid at 24 hours following a six hour wash was 0.054µg/cm² (0.765% of the dose applied). The proportion of the applied test substance that was washed off the skin at 6 hours was 85.1% with an additional 4.69% being removed by skin washing after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.322%), tape strips
1-2 (0.145%), tape strips 3-5 (0.412%) and 10.8% was found in the remaining skin. The mean total recovery was 102% of the dose applied.

The results obtained in this study demonstrate that the absorption of the test substance through human dermatomed skin following the application of the test substance/Azoxystrobin is slow and the vast majority of the test substance can be washed off the skin by normal decontamination procedures. These data predict that the dermal absorption of the test substance from potential exposure to this formulation concentrate and its aqueous spray strength dilutions would be generally low and will not exceed 12.12%.

Description of key information

- Oral absorption: 33.95 ± 4.95 %; OECD 417, Marciniszyn 1985 b

- Oral absorption: 20 %; excretion via faeces (main route) and in urine; extensively metabolised mainly via hydroxylation; OECD 417, Punler 2013

- Oral availability: 18 - 20 % of administered dose; estimated from the oral:iv urine excretion ratio; OECD 417, Hutton 2017

- Skin absorption: 12.12 % of administered dose; sum of reception fluid, exposed skin and skin strips concentrations; OECD 428, Noakes 2013

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

32

Absorption rate - dermal (%):

12.12

Absorption rate - inhalation (%):

100

Additional information

The current oral absorption value used to support the DNEL is 32% proposed, based on the Marciniszyn 1985a study. Several studies have been conducted in rat, where the fraction of dose absorbed is calculated to range from 20% (at 5 mg/kg po) to 32% (at 1.5 mg/kg po), based on the sum of administered dose in urine, bile, cage wash, tissues and carcass. The latest study by Hutton (2017), aimed to confirm the fraction of an oral dose systemically available by comparing it with an intravenous dose. The fraction of orally absorbed dose systemically available was estimated by the ratio of dose excreted in urine following oral dosing compared to intravenous dosing. In addition, the usually more predictable dose normalised blood AUC oral to intravenous ratio was used. The fraction of administered dose systemically available, based on the urinary excretion ratio (oral:iv) accounted for 18-19% of the administered dose. However, the blood data demonstrates an underestimation of oral bioavailability, ca 2%, where the fraction in urine is at least 5%. This is possibly due to effects of first pass metabolism, enterohepatic recirculation and/or the reactive nature of test substance in blood and tissues. As test substance is reactive and been shown to bind to blood, the introduction of test substance directly into the blood stream, causes the dose to behave very differently than if it enters via the GIT and liver. As the iv and oral dose appear to be handled physiologically differently via the different administration routes, the urine ratio data, can be used as supporting evidence, but is not definitive, and the blood data is clearly not representative. At much higher doses, well in excess of the NOAEL, the fraction of dose absorbed appears to be lower. This is a reflection of the solubility and dissolution characteristics of test substance in the GIT. As these are not realistic exposure scenarios, these values should be disregarded. Only the lower dose data should be considered, especially at the NOAEL, as this is the dose modified to generate the DNEL. What the intravenous route does show is that the systemically available dose is excreted via bile into faeces. Therefore, it is likely that the same happens for systemically available material following oral dosing. Therefore, the fraction of dose estimated in bile should be considered. After an oral dose of 5 mg/kg the fraction of an oral dose systemically available is estimated between 20 and 32%, based on the data by Marciniszyn 1985a and Punler et al 2013. Therefore, 32% remains the relevant value to be used for DNEL setting.

Dermal absorption - OECD 428 - Noakes, 2013

The absorption and distribution of test substance from a test substance/Azoxystrobin suspension concentrate (SC) formulation was measured in vitro through human dermatomed skin conforming to the OECD 428 testing guideline. The doses were applied to the dermatomed skin as the test substance/Azoxystrobin formulation concentrate containing 400 g test substance/L and three aqueous spray strength dilutions: nominally containing 10 g, 1.875 g and 0.667 g test substance/L: equivalent to 1/40, 1/213.3 and 1/600 w/v, respectively. The formulation and the aqueous spray strength dilutions were applied at rates of 10 µL/cm2 and left unoccluded for an exposure period of six hours and a total run time of 24 hours. The absorption process was followed by taking samples of the receptor fluid (50% ethanol in water) at recorded intervals throughout the exposure period. At the end of the experiment, the distribution of test substance in the test system was assessed, which included a tape stripping technique to determine its distribution in the stratum corneum and in the remaining skin.
The formulation concentrate was included to assess exposure to mixer/loaders. The spray strength dilutions used (10 g, 1.875 g and 0.667 g test substance/L) represented typical in use concentrations. These applications were designed to simulate potential human dermal exposure to the formulation during normal use.
The absorption and distribution were followed using [14C]-test substance which was incorporated into the doses prior to application. The samples were analysed by liquid scintillation counting (LSC).
The surface of the dermatomed skin was decontaminated after a six hour exposure period to investigate the amount of test substance absorbed by the end of a typical ‘working day’ period. After decontamination, the absorption of test substance was monitored for the remainder of the 24 hour observation period.

Absorption of test substance from the formulation suspension concentrate (400 g test substance/L) through human dermatomed skin was at a mean absorption rate of 0.008 µg/cm²/h during the 24 hour experimental period. The amount absorbed into the receptor fluid at 24 hours following a six hour wash was 0.206 µg/cm² (0.006% of the dose applied). The vast majority of the applied test substance (mean 105%) was washed off the skin at six hours, and an additional 0.238% was removed in the skin wash after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.039%), tape strips 1-2 (0.009%), tape strips 3-5 (0.007%) and 0.401% was found in the remaining skin. The mean total recovery was 105% of the dose applied.
The absorption rate of test substance from the 10 g/L aqueous dilution 1 through human dermatomed skin was 0.005µg/cm²/h during the 24 hour experimental period. The amount absorbed into the receptor fluid at 24 hours, following a six hour wash, was 0.120µg/cm² (0.119% of the dose applied). The majority of the applied test substance (mean 90.6%) was washed off the skin at six hours, and an additional 4.39% was removed in the skin wash after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.299%), tape strips
1-2 (0.181%), tape strips 3-5 (0.165%) and 7.89% was found in the remaining skin. The mean total recovery was 104% of the dose applied.

The absorption rate of test substance from the 1.875 g/L aqueous dilution 2 through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour experimental period. The amount
absorbed into the receptor fluid at 24 hours following a six hour wash was 0.056µg/cm² (0.285% of the dose applied). The proportion of the applied test substance that was washed off the skin at 6 hours was 99.7% with an additional 1.61% being removed by skin washing after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.076%), tape strips
1-2 (0.119%), tape strips 3-5 (0.182%) and 6.31% was found in the remaining skin. The mean total recovery was 108% of the dose applied.

The absorption rate of test substance from the 0.667 g/L aqueous dilution 3 through human dermatomed skin was 0.002 µg/cm²/h during the 24 hour experimental period. The amount absorbed into the receptor fluid at 24 hours following a six hour wash was 0.054µg/cm² (0.765% of the dose applied). The proportion of the applied test substance that was washed off the skin at 6 hours was 85.1% with an additional 4.69% being removed by skin washing after 24 hours. A small proportion of the dose applied was recovered from the donor chamber (0.322%), tape strips
1-2 (0.145%), tape strips 3-5 (0.412%) and 10.8% was found in the remaining skin. The mean total recovery was 102% of the dose applied.

The results obtained in this study demonstrate that the absorption of the test substance through human dermatomed skin following the application of the test substance/Azoxystrobin is slow and the vast majority of the test substance can be washed off the skin by normal decontamination procedures. These data predict that the dermal absorption of the test substance from potential exposure to this formulation concentrate and its aqueous spray strength dilutions would be generally low and will not exceed 12.12%.