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Administrative data

Link to relevant study record(s)

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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment.
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
The labelled compound was dosed to mice, rats, guinea-pigs, dogs, and rhesus monkeys. The absorption, distribution, metabolism and excretion of the test substance was studied.
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14C
Species:
other: mice, rats, guinea-pigs, dogs, and rhesus monkeys.
Strain:
not specified
Route of administration:
other: oral gavage or capsule /
Details on exposure:
The labelled test substance was dosed orally to small animals by catheter as an aqueous solution and to dogs and monkeys in capsules.
Duration and frequency of treatment / exposure:
Animals were dosed once
Dose / conc.:
1 other: mg/kg bw
Remarks:
Tissue distribution: Male guinea-pigs
Dose / conc.:
0.5 other: mg
Remarks:
Tissue distribution: Mice, oral and I.V. administration / equivalent to approximately 25 mg/kg bw
Dose / conc.:
1 other: mg/kg
Remarks:
Comparative serum levels: mice, rats, guinea pigs
Dose / conc.:
2.1 other: mg/kg bw
Remarks:
ADME: Rat
Dose / conc.:
1.7 other: mg/kg bw
Remarks:
ADME: Guinea-Pig
Dose / conc.:
12.9 other: mg/kg bw
Remarks:
ADME: Mouse
Dose / conc.:
8.4 other: mg/kg bw
Remarks:
ADME: Mouse
Dose / conc.:
0.25 other: mg/kg bw
Remarks:
ADME: Dog
Dose / conc.:
0.04 other: mg/kg bw
Remarks:
ADME: Rhesus Monkey
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (distribution): Urine, faeces, bile, blood and several organs (guinea pig: Liver, lung, heart, spleen, kidney, muscle, fat, and brain).
- Time and frequency of sampling (distribution), guinea-pigs: 1/4, 1/2, 3/4, 1, 1.5, 2, 3, 4, 6, 24, 48 hours after dosing
- Time and frequency of sampling (distribution), mice: 1/4 and 1 hour after oral dosing and 1/12, 1/4, 1/2, 1, 3, 5, and 24 hours after intravenous dosing
- Time and frequency of sampling (ADME), all species: 4, 5, 6, 7, 24, 31, 48, 55, 72, 96 hours (urine and faeces)

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, bile
- From how many animals: all animals/species
- Time and frequency of sampling (ADME), all species: 4, 5, 6, 7, 24, 31, 48, 55, 72, 96 hours (urine and faeces)
- Method type for identification: thin-layer chromatography (T.L.C.) in chloroform/methanol (9:1) on merck silica GF plates, NMR, mass spectrometry, DAD

COMPARATIVE SERUM LEVELS OF THE LABELLED TEST SUBSTANCE AND ITS METABOLITES
- Tissues and body fluids sampled: blood
- From how many animals: Eight male mice, five male rats, and five male guinea pigs were each dosed orally (1.0 mg/kg) with the test substance. The sera from the mice were pooled and the sera from the other ten animals examined separately.
- Time and frequency of sampling: Animals were all killed 1 hour after dosing and blood removed.
- Other: The serum samples were counted to determine the total radioactivity levels and aliquots extracted with chloroform. The extracts were counted and also examined by T.L.C. in order that the amounts of each labelled component present could be determined. The serum levels of the test substance and the hydroxylated metabolite were calculated. Duplicate serum samples from the guinea-pigs and rats, and the pooled sera from the mice were assayed by the method based on the fluorescence of the test substance.

Details on absorption:
ADME IN ALL SPECIES (SINGLE EXPOSURE)
The test substance is well absorbed after oral administration in all test species.

Details on distribution in tissues:
TISSUE DISTRIBUTION OF RADIOACTIVE MATERIAL, GUINEA-PIGS
- The specific activity of the labelled test substance was 15,060 dpm/µg. - Serum and tissue levels were comparatively steady over the period 1/4 to 4 hours after oral administration, the maximum levels occurring at about 1 hour.
- The half-life of radioactivity in serum is of the order of 4 hours. Higher levels of radioactivity occur in bile, liver, and kidney, but it should be noted that even at 6 hours the comparatively high level in bile corresponds to only ca. 8 µg/mL. The brain level has dropped by 6 hours and was not detectable at 24 hours. Only very low levels of radioactivity were present at 28 hours in all tissues examined. See Table 3 in ‘any other information on results incl. tables’ for details.

WHOLE-BODY AUTORADIOGRAPHY, MICE
- The specific activity of the labelled test substance was 6.8 µCi/mg. - Radioactivity was distributed throughout all the tissues. It was almost completely cleared from the animal at 24 hours.
- At 15 min the orally dosed animal had very high activity in the stomach contents with some labelling of urine and bile, but no activity was detected in either liver or kidney at this time. At 1 hour after an oral dose, much activity still remained in the stomach but some absorption had occurred. Activity was detected in all tissues with high concentrations in renal medulla, urine and bile.
- At 5 min. after an i.v. dose, all tissues, including brain, intestinal wall and urine, were labelled. A small amount of activity was present in the contents of the pyloric region of the stomach. Levels of activity in all tissues, except bile, duodenal contents and urine, decreased progressively over the first hour. At one hour the nasal secretions were also labelled. At 3 and 5 hours all tissues were still labelled with high concentrations in bile, duodenal contents, bladder contents and nasal secretions. Very little activity was present in fully formed faecal pellets. After 24 hours, very little activity remained in the animal but the bladder wall was labelled.
- Since only a small proportion of dosed radioactivity is recovered in mouse faeces after oral administration, and since after the i.v. dose there is evidence of extensive biliary excretion, re-absorption from the gut is indicated in this species.
Transfer type:
blood/brain barrier
Remarks:
Guinea-pigs / Mice
Observation:
not determined
Details on excretion:
EXCRETION
- The radioactivity was measured in urine and faeces and exhaled air of the dosed animals.
- 96 hours after dosing total excretion of radioactivity was 104.6, 90.0, 90.2, 90.9, 104, 100.6% in the rat, guinea-pig, mouse, mouse, dog, and rhesus monkey, respectively.

FAECAL AND BILIARY EXCRETION
- The rat is the only species studied in which a significant proportion (43%) of an oral dose is excreted in faeces. Homogenisation of rat faeces in methanol afforded an extract containing 13% of the faecal radioactivity. The extract was shown by T.L.C. to contain a component having the same T.L.C. properties as the test substance (23%) and unresolved polar compounds.
- The labelled test substance was dosed orally (1.3 mg/kg bw) to a male rat whose bile duct had been cannulated. In the first 6 hrs, 20% of the dose was excreted in bile and by 44 hours, 58% of the dose. At the same dose level a male guinea-pig passed only 2.5% in bile in a 5.5 hour experiment the bile was found to contain the labelled test substance (15%), the hydroxylated metabolite (6%) and polar material (79%).

See Table 1 in ‘any other information on results incl. tables’ for details.
Metabolites identified:
yes
Details on metabolites:
URINARY METABOLITES
- Urine samples from different species were found to contain the amounts of unchanged test substance and its hydroxylated metabolite. See Table 2 in ‘any other information on results incl. tables’ for details.

SPECIES DIFFERENCES
- Urine samples from all species showed only two major fractions when examined by T.L.C.: the hydroxylated metabolite and polar (origin spot) material. The latter probably contains conjugates, but these have not been identified.
- The rat produces a minor urinary metabolite (5% of the material in urine) whose molecular weight corresponds to that of the test substance with an additional oxygen atom and an additional acetyl group. Its fluorescence spectrum (Ex nλ Max 285 nM; Em nλ max 389 nm) is identical with that of N-acetyl test substance. This minor metabolite has not been observed in urine from other species. But trace amounts of a labelled compound having the same T.L.C. properties have been found in chloroform extracts of mouse and guinea-pig serum.
- The mouse excretes a significant proportion (9.2, 8.4, and 4.7% in 3 experiments) of an oral dose (8 to 14 mg/kg bw) in exhaled air and in this respect differs from rat (1.6%) and guinea pig (none detected). This could indicate dealkylation, but the dealkylated compound has not been detected in urine from animals dosed orally with the test substance.
- As rat, mouse, guinea-pig, dog and rhesus monkey all produce the hydroxylated metabolite as the single major non-conjugated urinary metabolite, these species have at least one major metabolic pathway in common.

PROPERTIES OF URINARY HYDROXYLATED METABOLITE
- The hydroxylated metabolite has been isolated from a chloroform extract of mouse urine. It has not been synthesised. The structure of the metabolite was deduced from its mass spectrum and N.M.R. spectrum. The parent ion in the mass spectrum has m/e = 223, showing that the molecular formula differs from that of test substance by one oxygen atom. The N.M.R. spectrum shows that all the protons of the propyl side chain and the ring proton are intact. However, the signal due to the methyl group in the test substance (8.04 T) is replaced by a two-proton signal at 5.76 T, chemical shift being consistent with substitution at the carbon by an oxygen function.
Bioaccessibility (or Bioavailability) testing results:
COMPARATIVE SERUM LEVELS OF THE LABELLED TEST SUBSTANCE AND ITS METABOLITES
- See any Table 4. in 'any other information on results incl. tables' for details.
- the total (radioactive) serum level in guinea-pig is about five to six times higher than those in rat and mouse.
- 96 % of the material in guinea-pig serum is extractable into chloroform at physiological pH, whereas 70 % is extractable from rat serum and only 55 % from mouse serum.
- A major component in the serum in all three species is the labelled test substance (guinea-pig 71 %, rat 57 %, mouse 34 %).
- The hydroxylated metabolite is a minor component in the serum of all three species (guinea-pig 5 %, rat 4 %, mouse 7 %).
- There is an excellent agreement in the two sets of results from the TLC method and the method based on the fluorescence of the test substance.See any Table 5. in 'any other information on results incl. tables' for details.

Table 1. Excretion of radioactivity following oral administration of the labelled test substance

Species

Rat (male)a

Guinea-Pig (male)b

Mouse (male)c

Mouse (male)d

Dog (male)

Rhesus monkey (female)

Dose mg/kg bw

2.1

1.7

12.9

8.4

0.25

0.04

Time (hr.)

U

F

U

F

U

F

U

F

U (only)

U (only)

4

 

 

 

 

 

 

 

 

 

37.7

5

 

 

 

 

 

 

 

 

 

51.3

6

 

 

 

 

 

 

 

 

 

58.5

7

23.3

2.6

11.8

0.6

15.3

 

21.8

 

 

65.7

24

55.0

21.5

63.5

1.8

66.3

9.1

67.7

 

100.6

 

31

57.3

 

69.0

 

 

 

70.6

 

 

98.6

48

59.4

41.6

73.6

11.8

69.6

10.6

74.4

 

103.9

100.4

55

 

 

74.1

 

 

 

75.0

 

104.0

100.6

72

59.7

42.9

74.8

14.3

70.0

11.0

76.9

5.6

104.0

 

96

59.8

43.2

75.0

15.0

 

 

 

 

 

 

Total (U + F)e

104.6

90.0

90.2

90.9

104.0

100.6

a. 1.6% dose in exhaled air in the first 24 hr.

b. No radioactivity detected in exhaled air.

c. 9.2% dose in exhaled air in the first 24 hr.

d. 8.4% dose in exhaled air in the first 24 hr.

e. Included % dose in exhaled air where appropriate.

U = Urine

F = Faeces

 

Table 2. Proportions of the test substance and its metabolite in urine

Species

Dose mg/kg bw

Sample (hr)

% Dose in sample

% test substancea

% hydoxylatedmetabolitea

Rat (male)

2.1

7 - 24

32

9.6

14.8

Guinea-pig (male)

1.7

7 - 24

52

2.9

36.8

Dog (male)

0.25

0 - 24

100

1.0

33.0

Rhesus Monkey (female)

0.04

0 - 4

38

3.0

38.1

Mouse (male)

13.5

0 - 7

44

2.3

35.2

a: of the total radioactivity in the urine sample indicated.

Table 3. Distribution of labelled material in guinea-pigs following administration of the labelled test substance 1.0 mg/kg

Time (hr)

Serum

Blood

Bile

Liver

Lung

Heart

Spleen

Kidney

Muscle

Fat

Brain

¼

16.4a

11.5

19.5

15.7

3.8

6.9

2.6

7.2

3.8

1.8

3.8

100b

70

119

96

23

42

16

44

23

11

23

½

16.0

11.7

24.2

13.0

5.7

3.9

3.0

6.6

4.3

2.2

3.6

100

73

151

81

36

24

19

41

27

14

22

¾

16.6

12.9

33.4

14.9

7.9

7.6

3.2

7.6

5.5

2.6

4.8

100

78

202

90

48

46

19

46

33

16

29

1

22.4

17.9

46.8

18.9

5.6

12.3

3.1

10.6

7.8

3.8

7.2

100

80

209

84

25

55

14

47

35

17

32

1 ½

21.1

14.4

47.8

16.4

6.0

11.2

3.1

11.1

8.2

3.2

4.0

100

68

226

77

29

53

14

53

39

15

19

2

21.4

14.7

57.7

16.5

6.4

8.5

2.9

11.2

11.2

2.9

4.8

100

69

269

77

30

40

13

52

52

13

22

3

17.2

clotted

69.8

15.4

5.1

4.9

3.1

12.3

10.3

3.4

4.9

100

406

90

30

29

18

72

60

20

28

4

17.5

11.6

78.2

14.2

6.5

7.7

2.9

10.7

5.4

2.9

4.2

100

66

446

81

37

44

16

61

31

16

24

6

11.6

8.6

122.5

10.0

4.5

3.5

2.8

11.0

5.0

2.2

2.9

100

74

1058

86

39

30

24

95

43

19

25

24

0.41

0.30

3.6

0.86

0.31

0.10

0.09

0.35

0.15

0.20

ND

100

71

863

207

75

24

22

85

35

47

48

0.23

0.15

0.60

0.28

0.20

0.08

ND

0.23

ND

ND

ND

100

60

256

119

85

32

98

a. Throughout dpm/mL or g x 10-3

b. Throughout % serum level

ND: not detected

Table 4. Comparative serum levels in guinea-pig, rat and mouse

Species (all male)

Serum level dpm/mLa

% extracted into chloroformb

% composition of chloroform extract determined by TLC (A) and calculated % in serum (B)

 

 

 

Test substance

Hydroxylated Metabolite

 

 

 

A

B

A

B

Mousec

2852

55

62

34

13

7

Rat (average n=5)d

3714 ± 75

70 ± 4

 

57 ± 3

 

4

Guinea-pig (average n=5)d

18436 ± 648

96 ± 2

 

71 ± 2

 

5

a. Specific activity 15060 dpm/µg (as test substance)

b. At physiological pH

c. Values for serum pooled from 8 animals

d. ± S.E.

ND: Not detected (less than 1%)

Table 5. Comparison of serum levels of the test substance and hydroxylated metabolite determined by fluorescence and thin-layer chromatography

Species (all male)

Serum concentration of the test substance and its hydroxylated metabolite

 

By fluorescence µg/mL

By T.L.C µg/mL

Guinea-pig

1

1.12

0.91

 

2

1.07

0.94

 

3

1.06

1.04

 

4

1.04

0.99

 

5

0.88

0.76

Rat

1

0.10

0.13

 

2

0.12

0.16

 

3

0.15

0.18

 

4

0.12

0.14

 

5

0.12

0.13

Mice (8 animals)

0.12

0.08

Conclusions:
Interpretation of results: low bioaccumulation potential based on study results
The test substance is well absorbed after oral administration in mice, rats, guinea-pigs, dogs, and rhesus monkeys and excreted via urine and faeces as the parent compound or as its non-conjugated hydroxylated metabolite. The biological half life in dogs is < 3 hours.
Executive summary:

The test substance is well absorbed after oral administration in all species except rats. At least 70% of the dosed radioactivity is passed in urine within 48 hours. Rat differs from the other species in passing a large proportion (43%) of an oral dose in faeces. It has been shown that biliary excretion is of major importance in this species and whole body autoradiography indicates that biliary excretion and reabsorption occurs in mice. The test substance was extensively metabolised in all species studied. One single non-conjugated major metabolite occurs in the urine of all species, a metabolite in which the methyl group of the test substance has been hydroxylated. The sera from rat, guinea-pig, and mouse contain only small (4 - 7% of the total radioactivity in serum) of this metabolite; the one major component present being the test substance. The tissue distribution curves in guinea-pigs show that maximum levels of radioactivity in serum and brain occur at about 1 hour after oral administration. The serum and tissue levels were found to be steady over the period 1/4 to 4 hours after dosing. 48 hours after oral administration of the labelled test substance to a guinea-pig, the serum level was found to be 1 % of the maximum level at 1 hour and only very low levels of radioactivity were detected in liver, kidney, bile, lung and heart. The maximum serum level of the test substance is higher in guinea-pigs (0.87 µg/mL) than in rat (0.17 µg/mL) and mouse (0.06 µg/mL) after an oral dose of 1 mg/kg bw.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Objective of study:
bioaccessibility (or bioavailability)
Qualifier:
no guideline followed
Principles of method if other than guideline:
Two beagles per sex/dose were dosed daily for 3 months with capsules containing 0.15, 0.5, and 1.5 mg/kg bw test substance. The fluorescent properties of the test substance were used to determine the serum levels with a spectrophotofluorimeter.
GLP compliance:
no
Radiolabelling:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
Diet: Usual stock diet.
Route of administration:
oral: capsule
Vehicle:
other: lactose, maize starch, magnesium stearate
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
Tablets test substance were placed in hard gelatin capsules. Tablet composition:
- 0.6 mg test substance, 91 mg lactose, 7.4 mg maize starch, and magnesium stearate 1.0 mg (total tablet weight of 100 mg)
- 5 mg test substance, 180 mg lactose, 13 mg maize starch, and magnesium stearate 2.0 mg (total tablet weight of 200 mg)
- 6 mg test substance, 180 mg lactose, 12 mg maize starch, and magnesium stearate 2.0 mg (total tablet weight of 200 mg)
Duration and frequency of treatment / exposure:
daily for 3 months
Dose / conc.:
0.5 mg/kg bw/day
Dose / conc.:
1.5 mg/kg bw/day
No. of animals per sex per dose / concentration:
2
Control animals:
yes, concurrent no treatment
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: after 41, 55 and 90 days of treatment; 0.5, 1, 2, 4, 6 and 24 hours after the last dose
- Method for identification: The test substance was quantitavely extracted from serum at physiological pH into chloroform, an aliquot of which was evaporated to dryness. The residue containing the compound was taken up in water for spectrophotofluorimetric analysis. Into a glass-stoppered test tube were placed 1 mL serum 1 mL water (± standard amounts of the test substance) and 12 mL chloroform. The compound was extracted by shaking the tube for 5 - 10 minutes. The tubes were centrifuged to separate the solvent layer and a 9 mL aliquot of the chloroform transferred to a clean glass-stoppered tube. The chloroform was evaporated to dryness under a stream of air, with the water bath temperature at approximately 40 - 50 °C. The residue was dissolved in 2 mL water for fluormetric analysis. Fluorescence was measured in an Aminco-Bowman Spectrophotofluorimeter with activation of fluorescence set at 301 nm and emission at 430 nm (wavelengths uncorrected).
- Limits of detection and quantification: The test substance is intensely fluorescent, the minimum detectable level being in the order of 0.001 µg/mL water. Recovery of the test substance from serum was quantitative (i.e.100 %) when added at concentrations up to 1.0 µg/mL. In the range 0.01 to 1.0 µg/mL serum , the error in replication was not greater than 6 %. This method allowed detection of the test substance down to levels of about 0.005 µg/mL serum.
- Method for identification (metabolite): A major metabolite of the test substance is the hydroxy-methyl compound of the test substance. This metabolite has a fluorescent spectrum almost identical to that of the test substance and can be extracted from serum under identical conditions. Using the labelled test substance, this metabolite has been detected in rat serum, but, of the total radioactivity in the sample, only 4% was present in this form compared with 57% of unchanged test substance and some 30% of non-extracted activity.
Bioaccessibility (or Bioavailability) testing results:
There was no significant difference in the serum concentrations in males and females, nor was there a difference in the levels observed at 41, 55 and 90 days. There was a linear relationship between dose and peak serum concentration (slope: peak concentration of 0.26 µg/mL per 1 mg/kg dose). The areas under the average serum concentration time curve in the 0 - 6 hr. period were 0.245, 0.562 and 1.778 µg hr/mL for the 0.15, 0.5 and 1.5 mg/kg bw doses respectively; again there was a linear dose-response relationship (slope an area of 1.18 µg hr/mL per 1 mg/kg dose). See Table in 'any other information on results incl. tables'. Accurate determination of the biological half-life was not possible from the data obtained, but in 4 cases values of 2.1, 2.6, 2.6, and 5.3 hr. were recorded.

Table. Analysis of dog serum concentrations *

Dose mg/kg bw

N**

µg/mL serum ± S.E. Hrs. after dose

 

 

½

1

2

4

6

24

0.15

8

0.036 ± 0.015

0.045 ± 0.015

0.042 ± 0.009

0.050 ± 0.009

0.030 ± 0.007

0.015 ± 0.007

0.50

8

0.054 ± 0.018

0.092 ± 0.029

0.109 ± 0.021

0.104 ± 0.022

0.094 ± 0.020

0.030 ± 0.010

1.50

8

0.108 ± 0.034

0.238 ± 0.080

0.333 ± 0.085

0.392 ± 0.033

0.262 ± 0.037

0.042 ± 0.006

*Males and females combined; data on days 41, 55 and 90 combined

**Number of dogs

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
The substance is absorbed after oral dosing and average serum concentrations in the 0 - 6 hr. period were 0.245, 0.562 and 1.778 µg/hr/mL for the 0.15, 0.5 and 1.5 mg/kg bw doses respectively. Accurate determination of the biological half-life was not possible from the data obtained, but in 4 cases values of 2.1, 2.6, 2.6, and 5.3 hr. were recorded.
Executive summary:

Two beagles per sex/dose were dosed daily for 3 months with capsules containing 0, 0.15, 0.5, and 1.5 mg/kg bw test substance and the fluorescent properties of the test substance were used to determine the serum levels with a spectrophotofluorimeter on day 41, 55, and 90. There was no significant difference in the serum concentrations in males and females, nor was there a difference in the levels observed at 41, 55 and 90 days. There was a linear relationship between dose and peak serum concentration (slope: peak concentration of 0.26 µg/mL per 1 mg/kg dose). The areas under the average serum concentration time curve in the 0 - 6 hr. period were 0.245, 0.562 and 1.778 µg hr/mL for the 0.15, 0.5 and 1.5 mg/kg bw doses, respectively, and again there was a linear dose-response relationship (slope an area of 1.18 µg hr/mL per 1 mg/kg dose.) Accurate determination of the biological half-life was not possible from the data obtained, but in 4 cases values of 2.1, 2.6, 2.6, and 5.3 hr. were recorded. There was no evidence to suggest that the serum concentrations significantly increased or decreased after prolonged administration.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Objective of study:
bioaccessibility (or bioavailability)
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were dosed daily for 52 and 92 days with 0, 0.25, 1.25, and 5 mg/kg test substance by oral gavage. In addition extra rats were given a single doses or 14 doses (once daily) for serum level estimations. The fluorescent properties of the test substance were used to determine the serum levels with a spectrophotofluorimeter.
GLP compliance:
no
Radiolabelling:
no
Species:
rat
Strain:
other: albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: between 100 - 144 g
- Housing: 5 to a cage
- Diet: standard pelleted diet, ad libitum
- Water: ad libitum
Route of administration:
oral: gavage
Vehicle:
other: nonylphenolethyleneoxide 0.1%, sodium salt of sulphated cetyl/oleyl alcohol mixture 0.1%, polyglyceryl ricinoleate 0.1 %, and water to 100 %
Details on exposure:
VEHICLE
The suspensions of 0.1 % (w/v), 0.025 % (w/v), and 0.005% (w/v) were prepared by ball milling the drug into a solution of nonylphenolethyleneoxide 0.1 %, sodium salt of sulphated cetyl/oleyl alcohol mixture 0.1 %, polyglyceryl ricinoleate 0.1 %, and water to 100 % .
Duration and frequency of treatment / exposure:
daily for 1, 14, 52, or 92 days
Dose / conc.:
0.25 mg/kg bw/day
Dose / conc.:
1.25 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
No. of animals per sex per dose / concentration:
0.25 mg/kg: 2 (1 dose), 2 (14 doses), 1 (52 doses), 1 (92 doses).
1.25 mg/kg: 2 (1 dose).
5.0 mg/kg: 2 (1 dose), 2 (14 doses), 1 (52 doses), 1 (92 doses).
Control animals:
yes, concurrent no treatment
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: after 52 and 90 days of treatment; 0.5, 1, 2, 3, 4, 5, 6, 7 and 24 hours after the last dose
- Assay method for the test substance in serum: The test substance was quantitavely extracted from serum at physiological pH into chloroform, an aliquot of which was evaporated to dryness. The residue containing the compound was taken up in water for spectrophotofluorimetric analysis. Into a glass-stoppered test tube were placed 1 mL serum 1 mL water (± standard amounts of the test substance) and 12 mL chloroform. The compound was extracted by shaking the tube for 5 - 10 minutes. The tubes were centrifuged to separate the solvent layer and a 9 mL aliquot of the chloroform transferred to a clean glass-stoppered tube. The chloroform was evaporated to dryness under a stream of air, with the water bath temperature at approximately 40 - 50 °C. The residue was dissolved in 2 mL water for fluormetric analysis. Fluorescence was measured in an Aminco-Bowman Spectrophotofluorimeter with activation of fluorescence set at 301 nm and emission at 430 nm (wavelengths uncorrected).
- Limits of detection and quantification: The test substance is intensely fluorescent, the minimum detectable level being in the order of 0.001 µg/mL water. Recovery of the test substance from serum was quantitative (i.e.100 %) when added at concentrations up to 1.0 µg/mL. In the range 0.01 to 1.0 µg/mL serum , the error in replication was not greater than 6 %. This method allowed detection of the test substance down to levels of about 0.005 µg/mL serum.
- Method for identification (metabolite): A major metabolite of the test substance is the hydroxy-methyl compound of the test substance. This metabolite has a fluorescent spectrum almost identical to that of the test substance and can be extracted from serum under identical conditions. Using the labelled test substance, this metabolite has been detected in rat serum, but, of the total radioactivity in the sample, only 4% was present in this form compared with 57% of unchanged test substance and some 30% of non-extracted activity.
Bioaccessibility (or Bioavailability) testing results:
Despite the variable nature of the results, no consistently significant effect of repeated dosing was observed either in increasing or decreasing the levels seen after a single dose, nor was there a difference in the levels of males and females. All the results for a given dose level, therefore, were combined. The data obtained approximated to a linear relationship between dose in the range 0.25 - 1.25 mg/kg bw and (a) peak serum concentrations (slope: peak concentration of 0.11 µg/mL per 1 mg/kg dose) and (b) area under the curve from 0 - 7 hr. (slope: area of 0.5 µg hr/mL per 1 mg/kg dose), but on increasing the dose to 5 mg/kg bw, neither the peak level nor the area under the curve increased accordingly. See Table in 'any other information on results incl. tables'. Determination of the biological half-life of the test substance in rat was not possible from the available data.

Table. Rat serum levels after one or more doses

Dose mg/kg

µg/mL serum ± S.E. Hrs. after dose

 

½

1

2

3

4

5

6

7

24

0.25 Mean ± S.E. (males and females) (1 – 92 doses) (n = 12)

0.031 ± 0.007

0.019 ± 0.005

0.022 ± 0.006

0.008 ± 0.003

0.013 ± 0.004

0.008 ± 0.003

0.018 ±0.0.010

< 0.005

0

1.25 Mean ± S.E. (males and females) (1 dose) (n = 4)

0.115 ± 0.007

0.138 ± 0.007

0.112 ± 0.009

0.089 ± 0.003

0.092 ± 0.006

0.101 ± 0.014

-

0.080 ± 0.005

0.063 ± 0.002

5.0 Mean ± S.E. (males and females) (1 – 92 doses)

(n = 12)

0.233 ± 0.023

0.196 ± 0.030

0.154 ± 0.022

0.170 ± 0.038

0.142 ± 0.023

0.102 ± 0.021

0.187 ± 0.036

0.070 ± 0.012

0.084 ± 0.017

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
The substance is absorbed after oral dosing. The data obtained approximated to a linear relationship between dose in the range 0.25 - 1.25 mg/kg and peak serum concentrations (slope: peak concentration of 0.11 µg/mL per 1 mg/kg dose) and area under the curve increased accordingly.
Executive summary:

Rats were dosed daily for 52 and 92 days with 0, 0.25, 1.25, and 5 mg/kg test substance by oral gavage. In addition extra rats were given a single doses or 14 doses (once daily) for serum level estimations. The fluorescent properties of the test substance were used to determine the serum levels with an spectrophotofluorimeter. The data obtained approximated to a linear relationship between dose in the range 0.25 - 1.25 mg/kg and peak serum concentrations (slope: peak concentration of 0.11 µg/mL per 1 mg/kg dose) and area under the curve increased accordingly. Determination of the biological half-life of the test substance in rat was not possible from the available data. There was no evidence to suggest that the serum concentrations significantly increased or decreased after prolonged administration.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study the following hypothesis was investigated: a serum level of 0.4 µg/L is associated with doses at which vomiting in dogs could be expected to occur. A male beagle was dosed i.v. on three separate occasions with single doses of 0.05, 0.10, and 0.20 mg/kg to obtain pharmokinetic data. Based on these data, different infusion rates were calculated that should give rise to the target plasma level of 0.4 µg/L. 3 male beagles were i.v. exposed to the test substance with different infusion rates reaching plasma levels of 0.4 µg/L after ca. 30, 60, and 120 minutes.
GLP compliance:
no
Species:
dog
Strain:
Beagle
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation, determination pharmacokinetics: 17.5 kg
- Weight at study initiation, perfusion study: 12, 12, and 13 kg
Route of administration:
intravenous
Vehicle:
physiological saline
Details on exposure:
- Determination pharmacokinetics: One male beagle was dosed i.v. on three separate occasions with single doses of 0.05, 0.10, and 0.20 mg/kg to obtain pharmokinetic data.
- Perfusion study: 3 male beagles were i.v. exposed to the test substance with different infusion rates at 12.35, 6.66, and 3.87 mg/hr to reach plasma levels of 0.4 µg/L after ca. 30, 60, and 120 minutes.
Duration and frequency of treatment / exposure:
- Perfusion study: 30, 60, 120 minutes
Dose / conc.:
0.05 other: mg/kg
Remarks:
Determination pharmacokinetics
Dose / conc.:
0.1 other: mg/kg
Remarks:
Determination pharmacokinetics
Dose / conc.:
0.2 other: mg/kg
Remarks:
Determination pharmacokinetics
Dose / conc.:
3.87 other: mg/hr
Remarks:
Perfusion study
Dose / conc.:
6.66 other: mg/hr
Remarks:
Perfusion study
Dose / conc.:
12.35 other: mg/hr
Remarks:
Perfusion study
No. of animals per sex per dose / concentration:
- Determination pharmacokinetics: 1 male
- Perfusion study: 3 males
Control animals:
no
Details on study design:
- Dose selection rationale: The infusion rates were determined based on data obtained in a preliminary toxicokinetic study.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Determination pharmacokinetics: Blood samples were withdrawn from the femoral vein at 2, 4, 8, 10, 15, 20, 30, and 45 minutes and at 1, 1.5, 2, 4, 6 hours after dosing
- Perfusion study: plasma levels were determined at 10, 20, 30 and 60 minutes for an infusion rate of 12.35 mg/hr, at 15, 30, 60, and 90 minutes for an infusion rate of 6.66 mg/hr and after 30, 60, 90, 120, 150 minutes for an infusion rate of 3.87 mg/hr.
Preliminary studies:
DETERMINATION PHARMACOKINETICS
Half-life: 2.30, 1.83, and 2.30 hours after dosing 0.05, 0.10 and 0.20 mg/kg respectively.
Elimination rate: 0.300, 0.379, 0.300 per hour after dosing 0.05, 0.10 and 0.20 mg/kg respectively.
Plasma concentration: 0.066, 0.105, 0.276 µg/mL after dosing 0.05, 0.10 and 0.20 mg/kg respectively.
Volume distribution: 13.3, 16.6, and 12.7 L after dosing 0.05, 0.10 and 0.20 mg/kg respectively.
Bioaccessibility (or Bioavailability) testing results:
The measured plasma concentrations targeting 0.4 µg/L were attained at 22, 60 (0.34 µg/L), and 90 minutes with an infusion rate of 12.35, 6.66, and 3.87 mg/hr, respectively.

Clinical signs:

Experiment 1, 12 kg male beagle, 30 minute infusion, 12.35 mg/hr:

After 10 minutes infusion excessive salivation and the heart-rate increased from 125 to 160 beats/min. At fifteen minutes the dog became agitated with panting and violent defaecation occurred. These symptoms persisted throughout the period of infusion. The dog did not vomit.

Experiment 2, 12 kg male beagle, 60 minute infusion, 6.66 mg/hr:

During the first 20 minutes of the infusion the dog was quiet. At 30 minutes the dog became restless, began panting and the heart rate increased from 125 to 165 beats per minute at 50 minutes. The dog vomited at 40 and 60 minutes and there was some salivation. The degree of salivation increased during the post-infusion period.

Experiment 3, 13 kg male beagle, 120 minute infusion, 3.87 mg/hr:

During the first 50 minutes of the infusion the dog was a little restless, probably not due to the test substance. After 60 minutes infusion the dog began panting and became agitated. Heart rate increased from 130 to 170 beats per minute during the 60 to 70 minute period. During the 90 to 120 minutes of the infusion period the dog became more settled with period of agitation and the heart rate returned to 130 beats per minute.

The rates of infusion were of the correct order to give plasma levels approaching approximately 0.4 µg/L at three different rates. The onset of emesis in dogs was observed following intravenous infusion of the test substance at different rates.

Executive summary:

A male beagle was dosed i.v. on three separate occasions with single doses of 0.05, 0.10, and 0.20 mg/kg bw to obtain pharmokinetic data. Based on these data, different infusion rates were calculated that should give rise to the target plasma level of 0.4 µg/L. Three male beagles were i.v. exposed to the test substance with different infusion rates of 12.35, 6.66, and 3.87 mg/hr reaching plasma levels of 0.4 µg/L after ca. 30, 60, and 120 minutes, respectively. Pharmokinetic data resulted in a half life of 2.30, 1.83, and 2.30 hours, an elimination rate of 0.300, 0.379, 0.300 per hour, plasma concentrations of 0.066, 0.105, 0.276 µg/mL, and a volume distribution: 13.3, 16.6, and 12.7 L after dosing 0.05, 0.10 and 0.20 mg/kg respectively. In the infusion study plasma concentrations targeting 0.4 µg/L were attained at 22, 60 (0.34 µg/L), and 90 minutes with an infusion rate of 12.35, 6.66, and 3.87 mg/hr, respectively.

Description of key information

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Additional information

Toxicokinetics

Physical chemical properties

The test substance is a solid with a molecular weight of 207.2 g/mol. The log Pow value is 0.49 at 20 °C and the solubility in water is 5.7 g/L. The substance is stable in water at pH 4, 7 and 9 at 25 °C. These physico-chemical properties of the substance will enable qualitative judgements of the toxicokinetic behaviour (Guidance on information requirements and chemical safety assessment Chapter R.7.c: Endpoint specific guidance, R.7.12 Guidance on Toxicokinetics).

Absorption:

GI absorption

Based on the molecular weight, the log Pow and the water solubility the substance is likely to be absorbed in the GI tract. Gastro-intestinal absorption is likely to be triggered by passage via passive diffusion through aqueous pores or carriage with the bulk passage of water, which is favoured for small (molecular weight < 200 g/mol is considered small, the test substance is with a molecular weight of 207.2 g/mol a little larger than favourable for passive diffusion), moderate log P, water soluble substances. This is confirmed by data from studies measuring blood plasma levels of the test substance in mice, rats, guinea-pigs, dogs and humans after oral administration (Case and Dunlop, 1977; Farrell, 1970a ; Bayliss, 1973).

A single dose of 4 mg; ca. 0.06 mg/kg bw resulted in plasma levels of 0.081, 0.041, 0.034 µg/mL in one volunteer and 0.045, 0.056, 0.044 µg/mL in another volunteer, 1, 2 and 3 hours after dosing, respectively (Bayliss, 1973). This confirms that the test substance is absorbed in the gastro-intestinal tract of the volunteers. Since the substance has an emetic effect in humans, studies performed with animals that have vomiting reflexes are the most relevant. Farrell (1970a) dosed beagles daily for 3 months with 0, 0.15, 0.5, and 1.5 mg/kg test substance. The fluorescent properties of the test substance were used to determine the serum levels with a spectrophotofluorimeter at different time points after oral dosing (see Table1).

 

Table 1. Analysis of Dog serum concentrations*

Dose mg/kg bw

N**

µg/mL serum ± S.E. Hrs. after dose

 

 

½

1

2

4

6

24

0.15

8

0.036 ± 0.015

0.045 ± 0.015

0.042 ± 0.009

0.050 ± 0.009

0.030 ± 0.007

0.015 ± 0.007

0.50

8

0.054 ± 0.018

0.092 ± 0.029

0.109 ± 0.021

0.104 ± 0.022

0.094 ± 0.020

0.030 ± 0.010

1.50

8

0.108 ± 0.034

0.238 ± 0.080

0.333 ± 0.085

0.392 ± 0.033

0.262 ± 0.037

0.042 ± 0.006

*Males and females combined; data on days 41, 55 and 90 combined

**Number of dogs

Based on the physical chemical properties of the test substance and the measured serum levels in several species it can be concluded that the test substance is absorbed in the gastro-intestinal tract after oral administration. It should be noted that absorption is limited by the emetic effect of the test substance. Emesis is a systemic effect and occurs as a reflex response to the rapid increase of cyclic AMP in the blood caused by inhibition of the enzyme phosphodiesterase. In monkeys and marmosets the maximum tolerated dose is in the range of 0.1 to 0.5 mg/kg bw. 2 mg/kg is a certain emetic dose in dogs and monkeys. Humans are most sensitive to the emetic effects and vomiting occurred at 2 – 8 mg per person (ca. 0.03 – 0.11 mg/kg bw). Furthermore, Wright et al. (1979) observed inhibition of gastric emptying in mice, rats and monkeys after oral and subcutaneous dosing of the test substance. In rats inhibition of gastric emptying was maximal at plasma concentration above 0.2 µg/mL irrespective of the dosing route. It can be concluded that the test substance is absorbed via the gastro-intestinal tract, however the dose available for absorption is limited by emesis. Additional potential inhibition of gastric emptying could limit absorption.

Dermal absorption

The substance is a solid with a water solubility of 5.7 g/L, a molecular weight of 207.2 g/mol and a log Pow of 0.49. Dermal absorption is favourable for uptake at a molecular weight of <100 g/mol. Based on the water solubility of 5.7 g/L and the molecular weight, dermal absorption in the stratum corneum is expected to be limited. A dermal acute toxicity test in rats with 2000 mg/kg test substance did not result in substance related clinical signs or mortality. Although the plasma levels are unknown after dermal exposure the absence of mortality at a dose of 2000 mg/kg test substance (Davison 1988) indicates limited absorption when this result is compared with other toxicity tests in rats.

The plasma levels of rats were determined in an oral repeated dose study. The average plasma levels 30 min after dosing 5 mg/kg were 0.233 ± 0.023 µg/L confirming uptake (Farrell, 1970a). No mortality was observed in this study. A single oral dose in rats resulted in a LD50 between 150 and 155 mg/kg bw (Farrell, 1970b) and between 100 and 150 mg/kg bw (Davison, 1988). No mortality was observed at doses of 100 mg/kg bw in both studies. Intravenous dosing resulted in a LD50 between 50 and 75 mg/kg bw (Farrell, 1970b). Comparing these data it can be assumed that a 100% absorption of the 2000 mg/kg dermal dose would have resulted in mortality. It is likely that the absorption via the skin is at least 2000/155 = 13 times less efficient compared to gastro-intestinal absorption in the rat. Therefore it can be concluded that dermal absorption is limited and the dermal absorption is set at 10% of oral absorption.

Respiratory absorption

The substance is a powder and dissolved as an additive in plant protection products that can be sprayed. Therefore the substance is available for inhalation as powder or aerosol. The log P of 0.49 favours absorption directly across the respiratory tract epithelium by passive diffusion. Furthermore, substances absorbed via the GI are likely to be absorbed when inhaled.

Distribution

The distribution of the labelled test substance after oral administration was investigated in guinea-pigs. The distribution of the labelled material was determined from dosing until 48 hours after dosing. The labelled test substance was determined in plasma, blood, bile, liver, lung, heart, spleen, kidney, muscle, fat and brain (see Table 2).

 

Table 2. Distribution of labelled material in guinea-pigs following administration of the labelled test substance 1.0 mg/kg

Time (hr)

Serum

Blood

Bile

Liver

Lung

Heart

Spleen

Kidney

Muscle

Fat

Brain

¼

16.4a

11.5

19.5

15.7

3.8

6.9

2.6

7.2

3.8

1.8

3.8

100b

70

119

96

23

42

16

44

23

11

23

 

½

16.0

11.7

24.2

13.0

5.7

3.9

3.0

6.6

4.3

2.2

3.6

100

73

151

81

36

24

19

41

27

14

22

 

¾

16.6

12.9

33.4

14.9

7.9

7.6

3.2

7.6

5.5

2.6

4.8

100

78

202

90

48

46

19

46

33

16

29

 

1

22.4

17.9

46.8

18.9

5.6

12.3

3.1

10.6

7.8

3.8

7.2

100

80

209

84

25

55

14

47

35

17

32

 

1 ½

21.1

14.4

47.8

16.4

6.0

11.2

3.1

11.1

8.2

3.2

4.0

100

68

226

77

29

53

14

53

39

15

19

 

2

21.4

14.7

57.7

16.5

6.4

8.5

2.9

11.2

11.2

2.9

4.8

100

69

269

77

30

40

13

52

52

13

22

 

3

17.2

clotted

69.8

15.4

5.1

4.9

3.1

12.3

10.3

3.4

4.9

100

406

90

30

29

18

72

60

20

28

 

 

4

17.5

11.6

78.2

14.2

6.5

7.7

2.9

10.7

5.4

2.9

4.2

100

66

446

81

37

44

16

61

31

16

24

 

6

11.6

8.6

122.5

10.0

4.5

3.5

2.8

11.0

5.0

2.2

2.9

100

74

1058

86

39

30

24

95

43

19

25

 

24

0.41

0.30

3.6

0.86

0.31

0.10

0.09

0.35

0.15

0.20

ND

100

71

863

207

75

24

22

85

35

47

 

 

48

0.23

0.15

0.60

0.28

0.20

0.08

ND

0.23

ND

ND

ND

100

60

256

119

85

32

98

 

 

 

 

 

Throughout dpm/mL or g x 10-3

Throughout % serum level

ND: not detected

Metabolism

The test substance was extensively metabolised in rat, guinea-pig, mice, dog and rhesus monkeys. One single non-conjugated major metabolite occurs in the urine of all species, a metabolite in which the methyl group of the test substance has been hydroxylated (see Table 3).

 

Table 3. Proportions of the test substance and its hydroxylated metabolite in urine:

Species

Dose mg/kg

Sample (hr)

% Dose in sample

% test substancea

% metabolitea

Rat (male)

2.1

7 - 24

32

9.6

14.8

Guinea-pig (male)

1.7

7 - 24

52

2.9

36.8

Dog (male)

0.25

0 - 24

100

1.0

33.0

Rhesus Monkey (female)

0.04

0 - 4

38

3.0

38.1

Mouse (male)

13.5

0 - 7

44

2.3

35.2

a: of the total radioactivity in the urine sample indicated.

The sera from rat, guinea-pig, and mouse contain only small (4 - 7% of the total radioactivity in serum) of this metabolite; the one major component present being the test substance (Farrell, 1970a).

Excretion

Farrell (1970a) found that the test substance as well as its metabolite, in which the methyl group of the test substance has been hydroxylated, were excreted by the rat, guinea pig, mouse, dog and rhesus monkey after oral administration (see Table 4)

Table 4. Excretion of radioactivity following oral administration of the labelled test substance:

Species

Rat (male)a

Guinea-Pig (male)b

Mouse (male)c

Mouse (male)d

Dog (male)

Rhesus monkey (female)

Dose mg/kg

2.1

1.7

12.9

8.4

0.25

0.04

Time (hr.)

U

F

U

F

U

F

U

F

U (only)

U (only)

4

 

 

 

 

 

 

 

 

 

37.7

5

 

 

 

 

 

 

 

 

 

51.3

6

 

 

 

 

 

 

 

 

 

58.5

7

23.3

2.6

11.8

0.6

15.3

 

21.8

 

 

65.7

24

55

21.5

63.5

1.8

66.3

9.1

67.7

 

100.6

 

31

57.3

 

69

 

 

 

70.6

 

 

98.6

48

59.4

41.6

73.6

11.8

69.6

10.6

74.4

 

103.9

100.4

55

 

 

74.1

 

 

 

75

 

104

100.6

72

59.7

42.9

74.8

14.3

70

11

76.9

5.6

104

 

96

59.8

43.2

75

15

 

 

 

 

 

 

Total (U + F)e

104.6

90

90.2

90.9

104

100.6

a. 1.6% dose in exhaled air in the first 24 hr.

b. No radioactivity detected in exhaled air.

c. 9.2% dose in exhaled air in the first 24 hr.

d. 8.4% dose in exhaled air in the first 24 hr.

e. Included % dose in exhaled air where appropriate.

U = Urine

F = Faeces

Case and Dunlop (1977) found that plasma concentration in beagles were 0.066, 0.105, and 0.276 µg/mL after intravenous dosing of 0.05, 0.10 and 0.20 mg/kg bw test substance, respectively. The volume distribution was 13.3, 16.6, and 12.7 L, the elimination rate was 0.300, 0.379, 0.300 per hour and this resulted in half-lives of 2.30, 1.83, and 2.30 hours after dosing 0.05, 0.10 and 0.20 mg/kg bw, respectively. The half-life in beagles is < 3 hours (Farrell 1970a). It can be concluded that the test substance is rapidly excreted.

Accumulation

The substance is water soluble and the log P is 0.49. Therefore it is unlikely that the substance will accumulate in tissues. Furthermore the substance is metabolized and the test substance and its metabolites are excreted relatively fast via the urine and faeces (Farrell, 1970a). It can be concluded that it is unlikely that the test substance accumulates.