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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
experimental phase begin: 2005-07-13; end: 2005-11-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was generated according to generally valid and accepted testing guidelines and performed according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Objective of study:
absorption
excretion
other: tissue distribution... (see attached file)
Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Qualifier:
according to
Guideline:
EU Method B.36 (Toxicokinetics)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 27 males and 19 females from Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 7 weeks of age at the start of dosing
- Weight at study initiation: not stated
- Fasting period before study: not stated
- Housing:
Upon arrival (13 July 2005), the rats were housed under quarantine conditions and checked for overt signs of ill health and anomalies. Serological
investigation of the microbiological status, conducted in random samples, demonstrated that the animals were in good health.
Until the start of dosing, the animals were housed 3-4 per cage (Macrolon type IV) on sawdust. During dosing and collection of excreta, they were
housed individually in Nalgene metabolism cages (Techniplast).
The animals were identified by a group letter and a unique animal number on the tail, which was used for sample identification. The cages were
identified by cage cards showing the study number, group letter and animal number.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days, including one day of simulated test conditions in the metabolism cages


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 ºC
- Humidity (%): at least 30% and not exceeding 70%
- Air changes (per hr): 9-11 air changes per hour
- Photoperiod (hrs dark / hrs light): a sequence of 12 hours light and 12 hours dark


Administration / exposure

Route of administration:
other: Intravenous injection, Intraperitoneal, Oral (gavage)
Vehicle:
other: The test substance (Sb2O3) was formulated as a suspension in 0.5% hydroxypropyl methylcellulose (w/v) and 0.1% aqueous polysorbate 80 (w/v).
Details on exposure:
Preparation of dose formulations:

IV (intravenous) dose solution:
- The reference substance (SbCl3) was formulated as a solution in sterile isotonic saline solution. The Sb concentration in the dose solution was
determined by ICP-AES in aliquots taken prior to and directly after dosing.

Oral and IP (intraperitoneal) dose formulations:
- The test substance (Sb2O3) was formulated as a suspension in 0.5% hydroxypropyl methylcellulose (w/v) and 0.1% aqueous polysorbate 80 (w/v).
The Sb concentration in the dose formulations was determined by ICP-AES in aliquots taken prior to and directly after dosing.

For group E two dose formulations were prepared. The first was used on day 1-7, the second on day 8-14. The dose formulations were gently stirred using a magnetic stirring bar and visually checked for homogeneity before each administration. The Sb concentration in the dose formulations was
checked by taking aliquots just before and directly after dosing. In addition, the vehicle used for IP and PO administration was analyzed for any
antimony present.
Doses / concentrations
Remarks:
Doses / Concentrations:
Dose administration:

IV (intravenous) administration:
- Intravenous injection of 5 ml/kg BW in the tail vein.

IP (intraperitoneal) administration:
- Intraperitoneal dose administered by injection in the abdomen at 10 ml/kg BW.

PO (oral) administration.
- Oral (gavage) dose of 10 ml/kg BW administered by stomach tube.

No. of animals per sex per dose:
The study comprised six groups of rats.
Four non-dosed animals of each sex were used for blank urine and faeces (24 h collection) and for blank tissue samples.
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of toxicity data.
- Rationale for animal assignment (if not random): randomised
Details on dosing and sampling:
Amount dosed:
- Before dose administration, the animals were weighed and the dosing amount calculated. The exact dose was determined retrospectively by the
difference in weight of the needle plus syringe or stomach tube before and after administration of the formulated test substance. For group E
(repeated dose), the administered dose was based on volume. To verify the proper dose administration, the doses were also weighed on day 1, 8 and 14. For each rat, the administered dose and time of dosing was recorded.


Sample collection and storage:
- Samples were collected. All weights were recorded to ensure a proper mass balance.

- Urine was collected at room temperature. The samples were weighed, transferred to storage vials and kept frozen (<-18ºC) until analysis.
- Faeces were collected at room temperature. The faeces were diluted with water and homogenized at the end of the collection period.
The homogenate was weighed, transferred to storage vials and kept frozen (<-18ºC) until analysis.
- Blood for blood kinetics was taken from the tail vein (vena sacralis media) by cutting the tip of the tail. Blood (100 µl) was collected in haematocrit
capillaries and directly transferred into weighed Eppendorf cups containing 400 µl isotonic saline. Terminal blood was taken from the abdominal
aorta and
collected in heparinized tubes. After removal of whole blood aliquots, plasma was prepared by centrifugation at approximately 1200xg. Whole blood
and the remaining plasma were transferred to storage vials and kept frozen (<-18ºC) until analysis. The blood cells were discarded.
- At the defined time points, the animals were killed by exsanguination after anaesthesia with CO2/O2, and tissues and organs removed, weighed and kept frozen (<-18ºC) until analysis. The residual carcass and GI-tract were directly digested with aqua regia and stored at room temperature until
analysis.
- At the end of the collection period, cages were rinsed thoroughly with water/Triton X-100 (100/1; v/v). Samples were kept at room temperature
until analysis.
- Each sample was identified by study number, animal number, of samples sample type, and sampling time and date. In addition, samples received a
unique serial number 6502/Xnnn, where X is the type of sample. A sampling list was used to describe the coupling to the original samples.

Results and discussion

Main ADME resultsopen allclose all
Type:
absorption
Results:
absorption of diantimony trioxide after acute oral exposure to diantimony trioxide particle suspension in this study was low; only 0.3% after administration of 100 mg/ kg bw and 0.05% after administration of 1000 mg/ kg bw
Type:
absorption
Results:
absorption of antimony after oral dosing of diantimony trioxide is a slow process, with a Cmax at approximately 24h, followed by an even slower elimination phase from blood
Type:
distribution
Results:
antimony undergoes significant distribution to most tissues as it binds to red blood cells. Highest levels were found in bone marrow and thyroidea, followed by ovaries, spleen, liver, lung, heart, femur and skin
Type:
excretion
Results:
The major part of diantimony trioxide is excreted via faeces but it is also excreted via urine.

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
The ability of antimony to reach bone marrow after a single oral low dose was around 8 times lower than in the intravenously dosed rats.
In the IP-treated animals the Sb content in bone marrow was approximately 40 times higher than in the bone marrow of the IV-dosed and oral high
dose rats and approximately 300 times higher than in the bone marrow of the oral low dose rats.
In the control group, although very low amounts of Sb was found in whole blood, significant and comparable concentrations of antimony were
measured in the bone marrow (80 and 142 ng antimony/ g tissue, males and females respectively) and thyroid (98 and 195 ng antimony/ g tissue,
males and females respectively). These concentrations were also comparable with the antimony concentrations found in the same organs of the oral
low dose group and were also similar to the blood concentration in these animals.
In the oral high dose group the antimony concentrations were again comparable in the bone marrow (1192 and 1996 ng antimony/g tissue, males
and females respectively) and thyroid (1507 and 2103 ng antimony/g tissue, males and females respectively) and 2-3 times higher than in whole
blood (708 and 640 ng antimony/g tissue, males and females respectively).
After repeated oral dosing the antimony concentration in bone marrow was 2 times higher and in whole blood 10 times higher than the
concentrations seen in the respective organs of the single oral high dose group.
The antimony concentration was, after either a single oral low or high dose in the other measured organs, in femur around 3 times higher, in kidney
around 5 times higher and in lung, liver and skin around 10 times higher than controls. In heart, spleen and ovaries the antimony concentration was
increased around 6, 5 and 2 times, respectively after a single oral low dose and around 10-15 times after a single oral high dose, compared with controls.
The concentration in femur, kidney, lung, liver, skin, heart, spleen and ovaries increased further with a factor of ten after repeated oral high dosing
compared with the single oral high dose.
Details on excretion:
In the rats intravenously dosed with SbCl3 (group A), around 30% of the total antimony was excreted in the faeces within the first 24hours and around 12% was recovered from the urine 24 h after dosing. Therefore, it can be concluded that at 24 hours, the biliary excretion of absorbed antimony is
higher than the amount of urinary excretion.
In the IP-dosed animals (group B), 36% of the antimony was recovered from the faeces 72hours after dosing. In the orally dosed animals around 80%
and 100% of the Sb was excreted via the faeces 72houes after dosing. Only very low amounts of antimony were found in the urine, the tissue and
carcass in both orally dosed groups. The urinary excretion of antimony in male and female rats after repeated oral dosing was monitored during the 14 days of the dosing period and for 3 days afterwards. The excretion to urine was relatively stable with some fluctuation during the exposure period.Excretion decreased after the last day of exposure.

Metabolite characterisation studies

Details on metabolites:
Antimony as an inorganic substance is not metabolised in antimals or humans.

Any other information on results incl. tables

Table 2: Concentration and homogeneity of dose formulations:

dose group

(day of preparation)

concentration (mg Sb/kg dose formulation)

before dosing

after dosing

mean

%CV

A

155.3

151.9

153.6

1.6

B

7972

8349

8161

3.3

C

8215

8635

8425

3.5

D

77310

77937

77623

0.6

E

75431

75316

75374

0.1

F

no dose

no dose

1

-

Animal observation and dose administration:

The animals were checked for appearance and behaviour during acclimatization, at the time of administration, and at each sampling time. No study- or test substance-related signs of toxicity or unusual behaviour were observed. Body weight gain was normal with no differences between the sexes from the various treatment groups.

Doses are expressed as mg Sb/kg body weight and as mg SbCl3/kg body weight (group A) or Sb2O3/kg body weight (group B-E). The administered dose was close to the intended dose for all animals.

One of the male rats receiving the repeated oral high dose (group D) died as a result of incorrect dosing (into to the lungs) at day 8. Some other animals of group D (nr 54, 58 an 51 had some dark red spots on the longs. Instillation of test substance can be excluded, since the Sb content of the longs from these animals was not elevated as compared to the other animals of this group.

At necropsy it was observed that animals that received the IP dose still had visible residues of test substance in the abdominal cavity and thus, absorption from the dosing site was not complete.

Blood kinetics:

The blood concentration of antimony versus time curve after intravenous injection (group A)  can be divided into two phases;

- first a rapid distribution phase from the blood into an extravascular compartment leading to the lowest concentration of antimony in blood, during the 72 h of observation, at 4 hours after injection.

- second, slow redistrution phase of antimony back into blood is observed with a maximal level of antimony being reached at 48 hours after injection.

A slight elimination from blood was observed at 72 hours. After oral administration (group C and D) the absorption from the gastrointestinal tract into the vascular system was slow with a Cmax at approx. 24 hours after dosing for both dose groups. Despite a 10 times difference in dosing, the Cmax was only 2 times higher in group D (1000 mg/kg) than group C (100 mg/kg). The difference between both dose groups for the calculated AUC (0 -72h) was in the same range with a ratio between the oral low and the high dose of 2.09 and 1.37 in males and females.

After Cmax was reached, elimination from blood was slow and decrease in blood concentration was only observed at 72 hours after both oral doses and IV administration.

During the 14 days repeated oral high dosing, antimony blood concentration increased almost linearly with time up to 24 hours after the last dose and then decreased slowly (5%/day) during the next two days. This indicates that a steady state level was not reached after 14 days exposure at the dose level tested.

The blood concentration versus time curve observed after IP injection (group B) presented no clear peak and no elimination phase for the mean of all animals. Individual data show that the blood concentration increased steadily in time for three animals but reached a maximum value at 72 hours for only two of four animals. At necropsy it was observed that animals that received the IP dose still had visible residues of the test substance in the abdominal cavity indicating a continuous release of antimony from the dosing site in the group B. The dosed antimony trioxide may only be slowly removed from the peritoneal cavity

Table 3: Blood kinetics of antimony trioxide

group

A

B

C

D

sex

male

male

male

female

male

female

dose

(mg Sb/kg BW)

0.752

86.8

87.3

87.2

865

852

Cmax

(ng/g)

1353

11380

400

287

769

680

Tmax

(h)

24

72

24

24

24

48

T1/2

(h)

(83)2

n.a.

(21)2

(46)2

(223)2

(253)2

AUC (0-72h)

(ng/g.h)

73900

309000

21200

28300

44400

38700

AUC(0-infinity)

(ng/g.h)

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

Bioavailability1

(%)

-

-

0.247

0.330

0.052

0.046

1 calculated only based on the area under the measured data (AUC 0-72h)

2 calculated only on the last two measured points and, thus, not reliable

n.a. : not applicable

Parameter /Unit /Description:

Cmax / (ng/g) / Observed maximal blood concentration.

Tmax / (h) / Time to reach Cmax

AUC(0 -n) / (ng/g*h) / Area under the blood concentration versus time curve up to the last sampling point using the log/linear trapezoidal rule.

AUC(0-infinity) / (ng/g*h) / Total area under the blood concentration curve, calculated as AUC(0-n).+ Cn/ k2 , where Cn is blood concentration at the last sampling point.

k2 / (h-1) / Elimination rate constant, calculated by linear regression analysis of the terminal part (Cmax to last time point) of the log-concentration versus time curve.

T1/2 terminal / (h) / Elimination half-life = ln(2)/ k2.

Bioavailability IP dose / (%) / AUC(0-infinity) [IP]/ AUC(0-infinity)[IV] * Dose [IV] / Dose [IP] * 100. Both doses were first converted to mg Sb/kg

Bioavailability PO dose / (%) / AUC(0 -infinity) [PO]/ AUC(0-infinity)[IV] * Dose [IV] / Dose [PO] * 100. Both doses were first converted to mg Sb/kg

Table 4: Recovery of antimony in excreta and tissues in the various single dose groups- Expressed as % of the dose.

Dose group

A

B

C

C

D

D

Dose

male

female

male

female

(mg Sb/kg bw)

0.752

86.1

87.3

87.2

865

852

Urine

0-24h

11.62

0.22

0.040

0.025

0.0066

0.0061

24-48h

1.57

0.10

0.003

0.006

0.0009

0.0013

48-72h

0.67

0.06

0.006

0.003

0.0004

0.0007

Subtotal

13.76

0.39

0.050

0.034

0.0079

0.0080

Feces

0-24h

31.8

34.13

71.70

73.37

98.38

90.83

24-48h

7.8

1.96

7.45

8.21

1.68

7.83

48-72h

9.1

0.18

0.09

1.75

0.01

0.07

Subtotal

n.a.

36.26

79.23

83.33

100.07

98.74

Cage wash

0-61

0.01

0.01

0.01

0.002

0.001

Total excreted

n.a

36.67

79.29

83.37

100.08

98.75

GI-tract

(including content)

n.a

n.a

0.004

0.019

0.002

0.003

Tissues residues

Excised*

n.a

n.a

0.007

0.008

0.0043

0.0023

Organs

n.a

n.a

0.002

0.002

0.0003

0.0004

Carcass

n.a

n.a

0.023

0.021

0.0017

0.0023

Total retained

n.a

n.a

0.033

0.031

0.0063

0.0051

Total recovery

n.a

n.a

79.33

83.42

100.09

98.76

Table 5: Tissue distribution of antimony (ng Sb/g tissue) after single or repeated dosing of diantimony trioxide in rats.

F

Control

A

Single IV

1.57 mg/kg SbCl3

B

Single IP

100 mg/kg

C

Single PO

100 mg/kg

D

Single PO

1000 mg/kg

E

14 d rep

1000 mg/kg

male

female

male

male

male

female

male

female

male

female

Whole blood

2.8

3.0

1111

11383

181

189

708

640

8278

6886

Plasma

1.8

2.8

3.1

18

3.1

3.2

2.8

2.6

21

10

Bone marrow

80

142

1083

40517

141

89

1192

1996

2486

3517

Femur

18.9

10.0

214

6081

56

38

48

32

254

265

Liver

3.9

3.0

n.d.

n.d.

36

25

41

64

823

675

Kidney

2.7

2.4

n.d.

n.d.

15

8

12

23

323

261

Lung

3.6

2.2

n.d.

n.d.

36

27

41

61

746

882

Heart

3.6

3.1

n.d.

n.d.

22

14

42

41

643

356

Spleen

10

32

n.d.

n.d.

83

50

197

113

1485

1386

Brain

1.4

1.4

n.d.

n.d.

1.4

1.4

2.2

1.4

30

17

Thyroid

98

195

n.d.

n.d.

158

120

1507

2103

2639

2280

Testes

0.8

n.a.

n.d.

n.d.

2.3

n.a.

2.8

n.a.

39

n.a.

Prostate

9.5

n.a.

n.d.

n.d.

11.4

n.a.

8.5

n.a.

80

n.a.

Uterus

n.a.

15.2

n.d.

n.d.

n.a.

12.8

n.a.

11.4

n.a.

116

Ovaries

n.a.

17.3

n.d.

n.d.

n.a.

29.4

n.a.

262

n.a.

665

Muscle

2.7

3.3

n.d.

n.d.

18

4.4

5.0

4.7

39

44

Skin

2.3

1.5

n.d.

n.d.

41

11

9.6

16

90

103

Residual Carcass

10.4

5.7

n.d.

n.d.

27

22

19

26

303

221

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: low absorption after oral exposure
In conclusion, the absorption of diantimony trioxide after acute oral exposure to diantimony trioxide particle suspension in this study was low; only 0.3% after administration of 100 mg/ kg bw and 0.05% after administration of 1000 mg/ kg bw.

From this study it can also be concluded that the absorption of antimony after oral dosing of diantimony trioxide is a slow process, with a Cmax at approximately 24h, followed by an even slower elimination phase from blood.

However, antimony undergoes significant distribution to most tissues as it binds to red blood cells. Highest levels were found in bone marrow and thyroidea, followed by ovaries, spleen, liver, lung, heart, femur and skin.

The major part of diantimony trioxide is excreted via faeces but it is also excreted via urine.