Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Repeated dose oral toxicity study of the test chemical
Author:
Ono et al
Year:
2015
Bibliographic source:
Fundamental Toxicological Sciences

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
Subacute repeated dose toxicity study was performed to determine the toxic nature of the test chemical orally in rats
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: β-bromostyrene
- Molecular formula: C8H7Br
- Molecular weight: 183.0473 g/mole
- Substance type: Organic
- Physical state: Yellow clear liquid
- Impurities: 0.4%
- Purity: 99.6%

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Remarks:
Sprague Dawley
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Atsugi Atsugi Breeding Center of Charles River Japan, Inc. (Kanagawa, Japan).
- Age at study initiation: 6 weeks
- Weight at study initiation: 182-216g for males and 145-171 g for females
- Fasting period before study: No data available
- Housing: Animals were individually housed in wire-mesh steel bracket cages (W 250 × D 350 × H 200 mm) and identified by using ear tags.
- Diet (e.g. ad libitum): Pellet diet (CRF -1,oriental yeast co., Tokyo, Japan), ad libitum
- Water (e.g. ad libitum): Tap water through bottle, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 degree C
- Humidity (%): 49-66%
- Air changes (per hr): 10-15 times/hr
- Photoperiod (hrs dark / hrs light): 12 hr/day (light on/off, 7:00/19:00).

IN-LIFE DATES: From: To :No data available

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
No data
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in corn oil to give a dose range of 0, 30, 125 or 500 mg/kg/day

DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food):No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 0, 30, 125 and 500 mg/kg/day
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): Lot no. WKJ3948
- Purity: No data available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
0, 30, 125 or 500 mg/kg/day
No. of animals per sex per dose:
Total: 72
0 mg/kg/day: 6 male, 6 female
30 mg/kg/day: 6 male, 6 female
125 mg/kg/day: 6 male, 6 female
500 mg/kg/day: 6 male, 6 female

For recovery period:
0 mg/kg/day: 6 male, 6 female
500 mg/kg/day: 6 male, 6 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose level were selected based on results obtained from 14-days range finding study using the same strains of rats. All males and females in the 1000 mg/kg died. Increases in relative liver and kidney weights were observed in 300 mg/kg group. Therefore, for the present study high dose was set at 500 mg/kg/day and middle and low doses was set at 125 and 30 mg/kg/day.

- Rationale for animal assignment (if not random): Stratified random sampling based on body weight
- Rationale for selecting satellite groups: 6 male and 6 female of 0 and 500 mg/kg/day dose group kept without treatment as a recovery group.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random):No data available
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2-3 times daily during dosing period and once daily during recovery period.
- Cage side observations checked in table [No.?] were included. Morbidity and mortality were observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the start of dosing and once a week during dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Before dosing and on days 1,4,7,10,14,17,21,24 and 28 for dosing periods and on days 1,3,7,10, and 14 for the recovery period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured on days 1,4,7,10,14,17,21,24 and 28 for dosing periods and on days 1,3,7,10, and 14 for the recovery period
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:week 4 for dosing period and week 2 of recovery period

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:No data
- Dose groups that were examined: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At day after end of dosing and recovery period
- Anaesthetic used for blood collection: Yes ,blood was collected from abdominal aorta under deep anesthesia
- Animals fasted: Yes, overnight fasting was performed
- How many animals: From all 72 animals
- Parameters checked in table [No.?] were examined.: red blood cell (RBC), Hemoglobin, hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin,platelets, prothrombin volume, WBC , lymphocytes, neutrophils, eosinophils,basophils,monocytes, large unstained cells and blood clotting parameters, such as prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen level (FIB) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of dosing period and at the end of recovery period
- Animals fasted: Yes, overnight fasting was performed
- How many animals: From all 72 animals
- Parameters checked in table [No.?] were examined.: Serum was analyzed for alkaline phosphatase (ALP), total cholesterol (T-CHO), triglyceride (TG), phospholipid (PL), total bilirubin (T-BIL), glucose (GLU), blood urea nitrogen (BUN), creatinine (CRNN), sodium (Na), potassium (K), chlorine (Cl), calcium (Ca), inorganic phosphorus (P), total protein (TP), albumin (ALB), and albumin/globulin (A/G) ratio. Plasma isolated from heparinized blood was analyzed for aspartate and alanine aminotransferases (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and γ-glutamyl transpeptidase (γ-GTP). Electrolytes were also analyzed

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was conducted during Week 4 in the dosing period and Week 2 in the recovery period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, 4 hrs fasting was performed with water ad libitum
- Parameters checked in table [No.?] were examined.: dipstick parameters, such as pH, proteins, ketone bodies, glucose, occult blood, bilirubin, urobilinogen, color, sediments, and volume. Urine volume and osmolality were also measured

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the dosing and recovery periods
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: functional observation including auditory, approach, touch, and tail pinch response, pupilary and aerial righting reflexes.in addition grip strengths were measured using CPU guage and motor activity was recorded at 10 min. interval for 1 hour.

OTHER:After blood collection
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals were sacrificed by exsanguination, and the organs and tissues of the whole body, including external surfaces, head, breast, and abdomen were observed macroscopically. The brain, adrenals, thymus, spleen, heart, liver, kidneys, testes, epididymides, ovaries, and uterus were removed and
weighed. In addition, relative organ weights were calculated from organ/body weight ratios.

HISTOPATHOLOGY: Yes, The cerebrum, cerebellum, spinal cord (chest), sciatic nerve, pituitary gland, thyroid, parathyroids, adrenal glands, thymus, spleen, submandibular lymph nodes, mesenteric lymph nodes, heart, trachea, lung (including bronchial), stomach, duodenum, jejunum, ileum (including Peyer's patches), cecum, colon, rectum, liver, kidneys, urinary bladder, testes, epididymides, prostate, ovaries, uterus, sternum (including bone marrow), femur (including bone marrow), and femoral skeletal muscle were fixed in 10% phosphate-buffered formalin. The eyeballs and optic nerves were fixed in phosphate-buffered 3 vol% glutaraldehyde/2.5 vol% formalin, and the testes and epididymides were fixed in Bouin’s solution.

Paraffin sections for microscopic examination were routinely prepared and stained with hematoxylin-eosin. In the control and high dose groups sacrificed at the end of the dosing period, all preserved organs were examined under a light microscope. If treatment-related histopathological changes were found, the same tissues were examined for low and middle dose groups and the recovery group.
Statistics:
Statistical analysis were performed by using Bartlett’s test for homogeneity of distribution for quantitative data in open field observation, functional observation and urinalysis, grip strengths, motor activity, body weight, food and water consumption, hematological and blood biochemistry findings, and organ weights. The Dunnett’s multiple comparison test and the Dunnett’s-type mean rank sum test were conducted for homogenous and non-homogenous distribution, respectively to compare the control and individual treatment groups. Parametric data obtained during or after the recovery period were analyzed by F-test for homogeneity of distribution. For comparison, the Student’s t-test and the Aspin-Welch’s t-test were conducted for homogenous and non-homogenous distribution.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Clinical signs and mortality:
Mortality: When treated with 500 mg/kg/day, One female rat found dead on Day 3. The cause of death was unclear.
Clinical signs: When treated with 500 mg/kg/day, Decrease in spontaneous movement on the first dosing day were observed in male and female rat as compared to control. However, no abnormalities were observed in the general conditions thereafter during the dosing period. No clinical signs were observed in any animal during the recovery period.

Body weight and weight gain: When treated with 125 mg/kg/day, significantly increased body weight were observed in female rat on days 17-24 during the dosing period but no significant differences were found in the 500 mg/kg groups throughout the study

Food consumption and compound intake
Food consumption: When treated with 500 mg/kg/day, significant decrease in food consumption at day 4 were observed in male and female rat in dosing period and in females at days 7 and 14 in recovery period was noted. When treated with 125 mg/kg/day, significant increased in food consumption at days 7-21 were observed in female rats as compared to control.

Compound intake: No data available

Water consumption and compound intake:
Water consumption: When treated with 125 mg/kg/day, significant decrease in water consumption was observed in female rats during Week 4 of the dosing period as compared to control. However, this change was not observed in the high dose group. No significant differences were seen with water consumption for either sex in the recovery group.

Compound intake: No data available

Opthalmoscopic examination: No data available

Haematology: Significant decreases in MCH were observed in males receiving 30 and 500 mg/kg, and significant decreases in MCH concentration were
observed in both sexes receiving 500 mg/kg at the end of dosing period. A significant increase in reticulocytes was also found in females receiving 500 mg/kg. However, these changes were slight, and no clear changes were observed in RBC or HGB. Other significant changes were a reduction in APTT and an increase in FIB in females receiving 125 mg/kg, but these changes were not observed at 500 mg/kg. However, these changes were determined to be incidental.

At the end of the recovery period, the only significant changes observed were a decrease in EOSs in males and an increase in MONOs in females. However, these changes were not observed at the end of the dosing period and hence these changes were determined to be incidental.

Clinical chemistry: At the end of the dosing period, T-CHO and PL levels were significantly increased in rats receiving 125 mg/kg and above, and TG levels were also significantly increased in females receiving 500 mg/kg. Slight but significant decreases were found in AST activity at doses of 125 and 500 mg/kg, in ALT activity at 500 mg/kg, and in LDH activity at 125 mg/kg in males. In the 500 mg/kg group, there was a significant increase in TP levels for either sex,
a significant increase in ALB levels in males, and a significant decrease in CRNN in males. Significant increases were observed in Cl in females and P in males receiving 500 mg/kg, as well as Ca in males receiving 125 and 500 mg/kg.

After the recovery period, TG levels remained significantly high in females in the 500 mg/kg group, but other changes observed at the end of the dosing period were not detected.

Urinanalysis: During Week 4 of the dosing period, significant increases in urine volume were observed in males receiving 125 and 500 mg/kg and in females receiving 500 mg/kg. A significant decrease in urine osmolality was also observed in males receiving 125 and 500 mg/kg. In the sediments, small round epithelial cells were observed in 5/12 males and 1/11 females receiving 500 mg/kg, and this change increased in males compared with the control group. No significant differences were seen in urine volume, osmolality, or qualitative measurements for either sex compared with the control groups during Week 2 of the recovery period.

Neurobehaviour:
Functional observations: No significant changes were observed in any parameter for either sex receiving the test substance during Week 4 of the dosing period. When treated with 500 mg/kg/day, significant decrease in landing foot splay was observed in male rats during week 2 of the recovery period. However, it was determined to be incidental because this sign was not observed during Week 4 of the dosing period.

Grip strength: A significant increase in hindlimb grip strength was observed in females receiving 125 mg/kg during Week 4 of the dosing period. However, this was not observed in the high dose group. A significant decrease in forelimb grip strength was observed in males receiving 500 mg/kg during Week 2 of the recovery period, but this change was not observed during Week 4 of the dosing period.

Motor activity: No significant change was observed in any male or female rats receiving the test substance during Week 4 of the dosing period. During Week 2 of the recovery period, a significant decrease was observed in males receiving 500 mg/kg only 40-50 min after the start of measurement.

Organ weights: When treated with 500 mg/kg/day, Significant increase in absolute and relative liver weight in male and female rats, significant increase in absolute and relative kidney weights and significant decrease in absolute testes weight in male rats and significant increase in relative kidney weights in female rats were observed as compared to control during dosing period. Significant increase in relative liver and heart weight was observed in female rats as compared to control at the end of recovery period.

When treated with 125 mg/kg/day, Significant increase in relative liver weight and decrease in brain weight were observed in female rats at the end of the recovery period.

When treated with 30 mg/kg/day, decreases in relative spleen weight were observed in male rats as compared to control at the end of the recovery period.

Gross pathology: When treated with 500 mg/kg/day, excess fluids in the abdominal and thoracic cavities, an enlarged liver, and dark red foci in the glandular stomach and unilateral small thyroids observed in female rat and enlargement of liver was observed in male and female rats at the end of dosing period.

Enlargement of liver was observed in male rats at the end of recovery period.

When treated with 125 mg/kg/day, dark red foci in the lung in one male rat and dark red foci in the glandular stomach in one female rat were observed at end of the dosing period.

When treated with 30 mg/kg/day, unilateral small thyroids in one female rat were observed at end of the dosing period.

Histopathology: When treated with 500 mg/kg/day, minimal to mild degree of eosinophilic bodies in tubular cells, Mild degeneration of renal tubular and minimal hyaline casts in kidneys, minimal to mild centrilobular hypertrophy of hepatocytes in liver and minimal hypertrophy of follicular cells in thyroids were observed in male rats and atrophy of Peyer’s patch in the ileum, dilation of the renal tubes with centrilobular necrosis and congestion in the liver, focal hemorrhage and accumulation of foamy cells in the lung, atrophy of the mesenteric and submandibular lymph nodes, an increase in hematopoiesis and atrophy of white pulp in the spleen, erosion in the glandular stomach, atrophy of the thymus, and a remnant of ultimobranchial bodies in the thyroid were observed in female rats as compared to control.

Minimal Tubular regeneration, mild to minimal Eosinophilic body in tubular cells interstitial mineralization Kidney and Hypertrophy of follicular cells in thyroid of male rat and Periportal vacuolation of hepatocytes in liver of female rat were observed at the recovery period.

When treated with 125 mg/kg/day, minimal to mild degree of eosinophilic bodies in tubular cells in kidney were observed in male rat and minimal hypertrophy of follicular cells in thyroids were observed in female rats as compared to control.

Other changes at the end of the dosing and/or recovery periods observed in the heart, rectum, kidneys, liver, prostate, skeletal muscle, spleen, stomach, thyroid, and urinary bladder were considered to be incidental findings due to the apparent situation or histopathological properties.

Effect levels

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect on survival, clinical sign, body weight, food consumption, Water consumption, haematology, clinical chemistry, urinanalysis, neurobehaviour, organ weights, gross pathology and histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Hematological values in the repeated dose 28-day oral toxicity study of β-bromostyrene in rats.

Dose(mg/kg/day)

At the end of the dosing period

At the end of the recovery period

0

30

125

500

0

500

Males

 

 

 

 

 

 

No. of animals

6

6

6

6

6

6

MCH (µg)

20.2 ± 0.5

19.3 ± 0.4*

19.6 ± 0.6

19.2 ± 0.6**

18.5 ± 0.4

18.5 ± 0.8

MCHC (g/dL)

36.8 ± 0.3

36.3 ± 0.3

36.4 ± 0.2

36.1 ± 0.6*

35.9 ± 0.4

35.9 ± 0.3

Differential leukocyte count (%)

 

 

 

 

 

 

EOS

0.7 ± 0.2

1.0 ± 0.5

0.8 ± 0.3

0.7 ± 0.2

1.4 ± 0.4

0. 9 ± 0.2**

Females

 

 

 

 

 

 

No. of animals

6

6

6

6

6

6

MCHC (g/dL)

37.3 ± 0.4

37.5 ± 0.6

37.2 ± 0.5

36.2 ± 0.6**

37.1 ± 0.4

36.8 ± 0.4

Reticulocyte (%)

1.2 ± 0.3

1.5 ± 0.3

1.4 ± 0.4

1.7 ± 0.2*

1.3 ± 0.4

1.2 ± 0.4

APTT (sec)

17.7 ± 1.6

16.2 ± 1.3

15.4 ± 1.4*

16.1 ± 1.8

18.2 ± 1.7

20.6 ± 2.0

FIB (mg/dL)

220 ± 19

234 ± 20

256 ± 12**

243 ± 18

214 ± 15

246 ± 40

Differential leukocyte count (%)

 

 

 

 

 

 

MONO

1.7 ± 0.7

2.0 ± 0.7

2.0 ± 0.9

2.2 ± 1.4

1.4 ± 0.3

2.3 ± 0.7*

Values are expressed as the mean ± standard deviation. *P < 0.05 and **P < 0.01 versus control.

 

Table 2. Clinical biochemistry values of the repeated dose 28-day oral toxicity study of β-bromostyrene in rats.

Dose(mg/kg/day)

At the end of the dosing period

At the end of the recovery period

0

30

125

500

0

500

Males

 

 

 

 

 

 

No. of animals

6

6

6

6

6

6

AST (IU/L)

65 ± 3

57 ± 4

56 ± 7*

57 ± 8*

66 ± 3

64 ± 6

ALT (IU/L)

28 ± 3

27 ± 3

24 ± 3

23 ± 4*

30 ± 3

27 ± 3

LDH (IU/L)

73 ± 19

60 ± 11

52 ± 8*

75 ± 17

51 ± 6

54 ± 6

CRNN (mg/dL)

0.25 ± 0.03

0.22 ± 0.03

0.22 ± 0.01

0.21 ± 0.03*

0.25 ± 0.02

0.25 ± 0.01

Ca (mg/dL)

9.5 ± 0.1

9.7 ± 0.3

9.9 ± 0.2*

9.9 ± 0.2**

9.7 ± 0.2

9.7 ± 0.2

P (mg/dL)

7.8 ± 0.4

8.4 ± 0.6

8.3 ± 0.3

9.7 ± 0.8**

7.4 ± 0.6

7.7 ± 0.3

TP (g/dL)

5.9 ± 0.1

6.0 ± 0.2

5.9 ± 0.2

6.4 ± 0.3**

6.3 ± 0.3

6.3 ± 0.3

ALB (g/dL)

2.7 ± 0.1

2.8 ± 0.1

2.8 ± 0.1

2.9 ± 0.1**

2.9 ± 0.1

2.9 ± 0.1

Females

 

 

 

 

 

 

No. of animals

6

6

6

6

6

6

T-CHO (mg/dL)

41 ± 7

53 ± 19

72 ± 19*

92 ± 18**

63 ± 8

83 ± 23

TG (mg/dL)

10 ± 4

14 ± 8

18 ± 6

19 ± 5*

17 ± 7

27 ± 8*

PL (mg/dL)

77 ± 11

91 ± 23

113 ± 23*

141 ± 23**

113 ± 16

137 ± 20

Cl (mmol/L)

113 ± 2

114 ± 1

114 ± 1

116 ± 1*

113 ± 1

111 ± 2

TP (g/dL)

6.1 ± 0.3

6.1 ± 0.2

6.1 ± 0.3

6.5 ± 0.2*

6.5 ± 0.3

6.7 ± 0.1

Values are expressed as the mean ± standard deviation. *P < 0.05 and **P < 0.01 versus control.

 

Table 3: Absolute and relative organ weights in the repeated dose 28-day oral toxicity study of β-bromostyrene in rats.

Item

Dose(mg/kg/day)

At the end of the dosing period

At the end of the recovery period

Males

Females

Males

Females

Absolute

(g)

Liver

Kidney (R + L)

Liver

Kidney (R + L)

Liver

Kidney (R + L)

Liver

Kidney (R + L)

0

10.50 ± 2.05

2.53 ± 0.25

6.26 ± 0.76

1.67 ± 0.15

11.80 ± 1.27

2.97 ± 0.37

6.29 ± 0.62

1.77 ± 0.11

30

12.02 ± 1.31

2.84 ± 0.33

6.69 ± 0.74

1.71 ± 0.21

-

-

-

-

125

12.08 ± 1.45

2.88 ± 0.25

7.42 ± 0.45*

1.91 ± 0.18

-

-

-

-

500

14.83 ± 1.49**

3.26 ± 0.34**

9.37 ± 0.79**

1.85 ± 0.13

12.06 ± 1.85

2.98 ± 0.26

7.38 ± 1.10

1.89 ± 0.28

Relative

(g/100 g BW)

0

2.94 ± 0.20

0.72 ± 0.07

2.83 ± 0.19

0.76 ± 0.04

2.83 ± 0.13

0.71 ± 0.05

2.55 ± 0.10

0.72 ± 0.04

30

3.26 ± 0.15

0.77 ± 0.05

2.96 ± 0.22

0.76 ± 0.09

-

-

-

-

125

3.32 ± 0.29*

0.79 ± 0.04

3.11 ± 0.22

0.80 ± 0.06

-

-

-

-

500

4.31 ± 0.24**

0.95 ± 0.08**

4.34 ± 0.23**

0.86 ± 0.07*

3.00 ± 0.22

0.75 ± 0.08

3.10 ± 0.31*

0.79 ± 0.09

Values are expressed as the mean ± standard deviation of six rats. *P < 0.05 and **P < 0.01 versus control.

BW, body weight; R, right; L, left.

 

Table 4. Histopathological findings in the repeated dose 28-day oral toxicity study of β-bromostyrene in rats at the end of the dosing period.

Organs

Findings

Sex

Males

Females

Dose (mg/kg/day)

0

30

125

500

0

30

125

500

Number

6

6

6

6

6

6

6

6

Heart

Focal myocarditis

Minimal

1

-

-

0

0

-

-

0

Intestine, rectum

Submucosal cell infiltration

Minimal

1

-

-

0

0

-

-

0

Kidney

 

 

 

 

 

 

 

 

 

Tubular regeneration

Minimal

2

3

2

3

1

-

-

2

Eosinophilic body in tubular cells

Minimal

0

0

2

0

0

-

-

0

Mild

0

0

0

6

0

-

-

0

Hyaline cast

Minimal

0

0

0

3

1

-

-

0

Interstitial mineralization

Minimal

1

5

4

4

4

-

-

2

Interstitial cell infiltration

Minimal

2

1

1

3

3

-

-

0

Tubular degeneration

Mild

0

0

0

2

0

-

-

0

Liver

 

 

 

 

 

 

 

 

 

Periportal vacuolation of hepatocytes

Minimal

1

0

0

0

1

2

1

1

Mild

0

0

0

0

3

2

0

1

Extramedullary hematopoiesis

Minimal

0

1

1

0

0

0

0

1

Granuloma

Minimal

0

0

0

0

0

0

0

1

Microgranuloma

Minimal

4

4

4

6

6

5

5

1

Mild

0

0

0

0

0

0

0

1

Central hypertrophy of hepatocytes

Minimal

0

0

0

5

0

0

0

5

Mild

0

0

0

1

0

0

0

1

Prostate

 

 

 

 

 

 

 

 

 

Lymphocyte infiltration

Minimal

3

-

-

5

-

-

-

-

Mild

1

-

-

0

-

-

-

-

Skeletal muscle

 

 

 

 

 

 

 

 

 

Cell infiltration

Minimal

1

-

-

0

0

-

-

0

Spleen

 

 

 

 

 

 

 

 

 

Increased hematopoiesis

Minimal

1

-

-

1

0

-

-

0

Glandular stomach

 

 

 

 

 

 

 

 

 

Erosion

Minimal

0

-

-

0

-

-

1 a)

1

Thyroid

 

 

 

 

 

 

 

 

 

Ectopic thymus

Minimal

2

0

0

0

0

0

0

0

Interstitial cell infiltration

Minimal

0

0

0

1

0

0

0

0

Remnant of ultimobranchial body

Minimal

2

1

2

1

2

2

0

3

Hypertrophy of follicular cells

Minimal

0

0

0

2

0

0

1

5

Urinary bladder

 

 

 

 

 

 

 

 

 

Submucosal cell infiltration

Minimal

0

-

-

1

0

-

-

0

-, not examined

a) Number of examined animals was one in which dark red foci was grossly observed in the glandular stomach.

 

Table 5. Histopathological findings in the repeated dose 28-day oral toxicity study of β-bromostyrene in rats at the end of the recovery period.

Organs

Findings

Sex

Males

Females

Dose (mg/kg/day)

0

500

0

500

Number

6

6

6

6

Kidney

 

 

 

 

 

Eosinophilic body in tubular cells

Minimal

0

3

-

-

Tubular regeneration

Minimal

0

2

-

-

Mild

0

3

-

-

Hyaline cast

Minimal

0

1

-

-

Interstitial mineralization

Minimal

3

5

-

-

Liver

 

 

 

 

 

Periportal vacuolation of hepatocytes

Minimal

1

0

1

2

Microgranuloma

Minimal

6

6

5

5

Thyroid

 

 

 

 

 

Ectopic thymus

Minimal

1

0

0

0

Remnant of ultimobranchial body

Minimal

2

1

0

1

Hypertrophy of follicular cells

Minimal

0

1

0

0

 -, not examined

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) was considered to be 30 mg/kg/day when Sprague Dawley [Crl:CD()SD] male and female rats were treated with the test chemical for 28 days.
Executive summary:

In a Subacute repeated dose oral toxicity study, Sprague Dawley [Crl:CD()SD] male and female rats treated with the test chemical orally by gavage in the concentration of 0 (corn oil), 30, 125 and 500 mg/kg/day for 28 days. Recovery group animals for a duration of 14 days were also included in the study other than the test group animals. The animals were observed for clinical signs, mortality, changes in body weight, food and water consumption, urinanalysis, hematology, clinical chemistry, neurobehavioral parameters were noted and the animals were sacrificed for gross and histo-pathology

One female rat was found dead on Day 3 and Decreases in spontaneous movement during weeks 3 and 4 were observed in male and female rats. No clinical signs were observed in any animal during the recovery period. Significantly increased body weight was observed in female rat on days 17-24 during the dosing period and significant decrease in water consumption was observed in female rats during week 4 of the dosing period a 125 mg/kg/day dose group as compared to control. Significant decreased in food consumption at day 4 were observed in male and female rat in dosing period and in females at days 7 and 14 in recovery period at 500 mg/kg/day and significant increased in food consumption at days 7-21 were observed in female rats at 125 mg/kg/day as compared to control. Decreased mean corpuscular hemoglobin (MCH) during dosing and eosinophils in recovery period were observed in male rat and increased reticulocyte and decreased mean corpuscular hemoglobin concentration (MCHC) during dosing and increase monocytes at recovery period were observed in female rats and significant increased in total protein level in male and female rats, Ca, P and albumin level and decrease in aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase and creatinine in male rats and significant increased in total cholesterol, triglycerides, phospholipids and Cl level in female rats at the end of dosing. Significant increased in triglycerides level was observed in female rats after the recovery period at 500 mg/kg/day. Decreased activated partial thromboplastin time and increased fibrinogen level were observed in female rats andsignificant decrease in aspartate aminotransferase, lactate dehydrogenase level and significant increased in Ca level in male rats and significant increased in total cholesterol, phospholipids level in female rats observed at 125 mg/kg/day as compared to control. Significant increases in urine volume in male and female rats, decrease in urine osmolality and small round epithelial cells in sediments were observed in male rats during week 4 of dosing period at 500 mg/kg/day and significant increases in urine volume and significant decrease in urine osmolality were observed in male rats at 125 mg/lg/day as compared to control. No significant differences were seen in urine volume, osmolality and qualitative measurements for either sex compared with the control groups during week 2 of the recovery period. Significant decrease in landing foot splay and forelimb grip strength was observed in male rats during week 2 of the recovery period. However, it was determined to be incidental because this sign was not observed during week 4 of the dosing period at 500 mg/kg/day. Significant decrease in forelimb grip strength was observed in male rats during week 2 of recovery period. No significant change was observed in any male and female rats receiving the test substance during week 4 of the dosing period. Significant increase in hindlimb grip strength was observed in female rats during week 4 of the dosing period at 125 mg/kg/day as compared to control. However, this was not observed in the high dose group. Similarly, Significant increase in absolute and relative liver weight in male and female rats, significant increase in absolute and relative kidney weights and significant decrease in absolute testes weight in male rats and significant increase in relative kidney weights in female rats in dosing period and significant increase in relative liver and heart weight was observed in female rats at 500 mg/kg/day as compared to control at the end of recovery period. Significant increase in relative liver weight and decrease in brain weight were observed at 125 mg/kg/day in female rats at the end of the recovery period. In addition, excess fluids in the abdominal and thoracic cavities, an enlarged liver, and dark red foci in the glandular stomach and unilateral small thyroids observed in female rat and enlargement of liver was observed in male rats at the end of dosing period. Enlargement of liver was observed in male rats at the end of recovery period in 500 mg/lg/day. Dark red foci in lung in one male rat and dark red foci in the glandular stomach in one female rat were observed at end of the dosing period at 125 mg/kg/day dose group. Minimal to mild degree of eosinophilic bodies in tubular cells, Mild degeneration of renal tubular and minimal hyaline casts in kidneys, minimal to mild centrilobular hypertrophy of hepatocytes in liver and minimal hypertrophy of follicular cells in thyroids were observed in male rats and atrophy of Peyer’s patch in the ileum, dilation of the renal tubes with centrilobular necrosis and congestion in the liver, focal hemorrhage and accumulation of foamy cells in the lung, atrophy of the mesenteric and submandibular lymph nodes, an increase in hematopoiesis and atrophy of white pulp in the spleen, erosion in the glandular stomach, atrophy of the thymus, and a remnant of ultimobranchial bodies in the thyroid were observed in female rats at 500 mg/kg/day as compared to control. Minimal Tubular regeneration, mild to minimal Eosinophilic body in tubular cells interstitial mineralization Kidney and Hypertrophy of follicular cells in thyroid of male rat and Periportal vacuolation of hepatocytes in liver of female rat were observed at the recovery period. Minimal to mild degree of eosinophilic bodies in tubular cells in kidney was observed in male rat and minimal hypertrophy of follicular cells in thyroids was observed in female rats at 125 mg/kg/day as compared to control.

Therefore, the No Observed Adverse Effect Level (NOAEL) was considered to be 30 mg/kg/day when Sprague Dawley [Crl:CD()SD] male and female rats were treated with the test chemical for 28 days.