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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was generated according to generally valid testing guidelines.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Guideline:
other: no guideline specified, but conducted according to OECD 408.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 12 male and 12 female Wistar rats of the Alpk:APSD strain
- Housing: in multiple rat racks at four or five per cage initially and then in fours after they had been assigned to experimental groups
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 - 2 weeks
- no other details on test animals are stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 2°C
- Humidity (%): 55 +- 15%
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours per day fluorescent light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diets containing antimony trioxide
Details on oral exposure:
VEHICLE
- Concentration in vehicle: diets containing 0, 1000, 5000 or 20000 ppm antimony trioxide
- Purity: the homogeneity of the mixture was > 95%.
- no other details on exposure are reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity of antimony trioxide in the diet was determined by analysis of samples from the low and high dose levels. Samples were taken from
three separate levels in the mixer. Variation between samples was <5%. The chemical stability of antimony trioxide in the diet has been determined at
these dose levels for a period of up to 98 days.
Duration of treatment / exposure:
a 90-day duration study
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
20000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
12 male and 12 female Wistar rats
Control animals:
yes

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-side observations were made daily, which included recording changes in clinical condition or behaviour.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: More detailed clinical observations were made each time the body weight was recorded.


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each rat was recorded before exposure started and then once a week until termination.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded continously throughout the study for each cage of rats and calculated as a weekly mean.
- Received dose was calculated from the mean body weight for the inclusion rate of antimony trioxide.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination, blood samples were taken and analysed for haematology parameters.
- Anaesthetic used for blood collection: At termination of the study, rats were killed by an overdose of halothane and bled by cardiac puncture.
- Animals fasted: not stated
- Parameters checked: red cell count, haematrocrit, haemoglobin, mean cell volume, total and differential white cell count and platelet count.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination, blood samples were taken and analysed for clinical chemistry parameters.
- Animals fasted: No data
- Parameters checked: urea, glucose, total protein, albumin, cholesterol, triglycerides, total bilirubin, creatinine, sodium, potassium, chloride, calcium and phosphorus. The activities of alkaline phosphatase, alanine aminotransferase, gamma-glutamyl transferase, creatinine kinase and aspartate aminotransferase were also assessed using automated methods.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected over a 16 hours period from rats housed individually in metabolism cages during the final week of study.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: not stated
- Parameters checked : Volume, appearance, specific gravity and pH were measured for each urin sample, with semi-quantitative determinations of glucose, ketoses, bilirubin, protein and blood. An aliquote was centrifuged and the sediment was stained and examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes :
- Complete necropsies were performed on all rats.
- The adrenal glands, brain, kidneys, liver, epididymides and testes were weighed.
- All organs and tissues were examined for macroscopic lesions and fixed in 10% neutral buffered formalin or other appropriate fixative.

HISTOPATHOLOGY: Yes :
- All tissues from the controls and the top dose group were examined under the light microscope, together with any macroscopically abnormal tissue from the intermediate groups.
Statistics:
All data were evaluated using analysis of variance and/or covariance for each specific parameter using GLM procedure in SAS.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- There was no substance-related effect on clinical signs of toxicity.

BODY WEIGHT AND WEIGHT GAIN
- There was no substance-related effect on body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- There was no substance-related effect on food intake.
- Food consumptions was similar for all groups.

FOOD EFFICIENCY


HAEMATOLOGY
- The red cell count was slightly elevated for males, with a small (not statistically significant) decrease in mean cell volume; other red cell parameters were unaffected.
- Females showed a slight decrease in mean cell volume and a small (not statistically significant) increase in red cell count.
- White cell count and platelet count were unaffected in either sex.
- Three of the twelve (25%) males in the high dose group had slight (n=2) or moderate (n=1) plasma cell infiltration in the cervical lymph node.
- This was not observed in treated females or in any control animal.

CLINICAL CHEMISTRY
- changes in some clinical chemistry parameters :
- Male animals in the high dose group showed a 30% increase in triglycerides (P<0.01) and a 12% decrease in alkaline phosphatase (P<0.05).
- Alkaline phosphatase was also decreased in female animals both at 5000 (24%) and 20000 (37%) ppm (P<0.01) and in a dose-dependent manner.
- Cholesterol and aspartate aminotransferase levels were significantly increased in females in the high dose group by 13 and 52%, respectively.

URINALYSIS
- Analysis of collected urine showed a significant increase in volume and an accompanying decrease in specific gravity for females at the highest
dose of antimony trioxide. Minor intergroup pH differnces in male were not dose related and were considered incidental.

ORGAN WEIGHTS
- A 10% increase in absolute and relative liver weight was observed in female and male animals in the high dose group compared with controls.
- There were no effects on other organ weights at any dose level.


GROSS PATHOLOGY
- There was no gross findings at necropsy considered to be related to feeding antimony trioxide.


HISTOPATHOLOGY: NON-NEOPLASTIC
- no histological changes in the liver were observed to support an adverse effect on liver
- There was a slight increase in cysts in the pituitary of both sexes in the high dose groups. This was not considered to be treatment related.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 686 other: mg/kg bw/day
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
1 879 other: mg/kg bw/day
Sex:
female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

All rats survived the dosing period in good condition. The few clinical signs that were recorded can be considered to be more of an adaptive nature than typical for age and strain. None of these were attributed by the authors of the study to the presence of antimony trioxide in the diet.

Table 1: Survival, weight gain and dose received for rats fed diets containing antimony trioxide.

Mean body weight

Dose (ppm)

Survival

Initial

Final

Body weight gain (g)

Calculated mean dose (mg/kg/day)

Males

0

12 / 12

158 ± 16

488 ± 40

330

0

1000

12 / 12

159 ± 18

502 ± 64

343

84

5000

12 / 12

158 ± 19

487 ± 35

329

421

20000

12 / 12

159 ± 21

491 ± 28

332

1686

Females

0

12 / 12

139 ± 11

269 ± 12

130

0

1000

12 / 12

138 ± 12

266 ± 13

128

97

5000

12 / 12

139 ± 12

267 ±200

128

494

20000

12 / 12

142 ± 15

279 ± 16

137

1879

Values are means ± SD, based on 12 values; no significant differences at P0.05.

Table 2: Intergroup comparison of urinary parameters

dose (ppm)

volume (ml)

specific gravity

pH

males

0

7.4

1.040 ± 0.007

6.58 ± 0.51

1000

8.1

1.037 ± 0.005

6.92 ± 0.29*

5000

7.9

1.040 ± 0.006

6.75 ± 0.45

20000

7.7

1.038 ± 0.006

6.92 ± 0.29*

females

0

3.8

1.047 ± 0.012

5.92 ± 0.29

1000

4.5

1.044 ± 0.012

6.00 ± 0.00

5000

4.3

1.052 ± 0.019

5.75 ± 0.62

20000

6.8

1.036 ± 0.006**

6.17 ± 0.39

Results are mean ± SD (12 values); *P0.05 and **P0.01 compared to control value.

Table 3: Selected haematology parameters for rats fed diets containing antimony trioxide

Dose

(ppm)

Red cell count

(x10-12/L)

Haemoglobin (g/dl)

Haematrocrit

mean cell volume (fl)

White cell count

(x10-9/L)

Platelet count

(x10-9/L)

males

0

8.37 ± 0.33

15.2 ± 0.5

0.44 ± 0.02

50.2 ± 0.8

5.75 ± 1.13

801 ± 75

1000

8.78 ± 0.46

15.2 ± 0.6

0.44 ± 0.02

50.1 ± 1.1

6.48 ± 1.64

786 ± 107

5000

8.59 ± 0.43

14.8 ± 0.5

0.43 ± 0.02

49.7 ± 1.1

5.93 ± 1.08

792 ± 66

20000

9.09 ± 0.35*

15.5 ± 0.5

0.45 ± 0.02

49.2 ± 1.1

6.28 ± 1.42

802 ± 77

females

0

8.13 ± 0.36

15.1 ± 0.7

0.43 ± 0.02

53.3 ± 1.6

4.82 ± 1.17

769 ± 113

1000

8.23 ± 0.33

15.2 ± 0.4

0.43 ± 0.02

52.5 ± 1.2

4.83 ± 1.31

742 ± 81

5000

8.25 ± 0.42

15.2 ± 0.8

0.43 ± 0.03

52.3 ± 1.3

4.59 ± 1.29

755 ± 81

20000

8.30 ± 0.30

15.3 ± 0.5

0.43 ± 0.01

52.2 ± 1.3*

5.17 ± 1.07

774 ± 83

Results are means ± SD (12 values); *P0.05 compared to control value

Table 4: Selected clinical chemistry (plasma) parameters for rats fed diets containing antimony trioxide

Dose

(ppm)

Cholesterol

(mmol/L)

Triglycerides (mmol/L)

Alkaline phosphatase activity

(IU/L)

Alanine aminotransferase activity

(IU/L)

Aspartase aminotransferase

(IU/L)

males

0

2.23 ± 0.29

1.18 ± 0.24

202 ± 23

63.3 ± 11.2

91.7 ± 16.3

1000

2.20 ± 0.29

1.34 ± 0.22

220 ± 27

70.9 ± 13.5

96.9 ± 9.0

5000

2.29 ± 0.25

1.27 ± 0.20

197 ± 21

69.1 ± 10.4

99.6 ± 13.4

20000

2.39 ± 0.24

1.53 ± 0.40**

178 ± 42*

73.7 ± 34.8

112.2 ± 34.5

females

0

1.97 ± 0.32

0.70 ± 0.19

136 ± 40

46.6 ± 8.5

91.5 ± 21.6

1000

2.02 ± 0.28

0.75 ± 0.18

122 ± 22

48.6 ± 11.6

104.8 ± 33.8

5000

2.13 ± 0.27

0.81 ± 0.16

105 ± 17**

51.8 ± 17.7

100.1 ± 28.8

20000

2.22 ± 0.17*

0.76 ± 0.24

87 ± 14**

66.8 ± 56.7

138.8 ± 90.0**

Results are means ± SD (12 values); *P0.05 and **P0.01 compared to control value.

Applicant's summary and conclusion

Conclusions:
Two repeated dose oral studies suggest that diantimony trioxide may be toxic to the liver. This is based on a 10% increase in liver weight and significantly elevated ASAT values, although significantly decreased ALP values, in one study (Hext et al., 1999), supported by significantly elevated ASAT and ALP levels observed in another study (Sunagawa, 1981). However, in the absence of histological change or any clinical signs of antimony intoxication to support that the liver findings are adverse, the findings are regarded as adaptive or incidental to treatment with diantimony trioxide. A NOAEL of 1686 mg/kg/d for liver toxicity is suggested.