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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002 - 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: 875/318/EEC; 83/571/EEC; 91/507/EEC
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name of test material (as cited in study report): ATBC
Substance type: pure active
Physical state: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
12 months
Frequency of treatment:
Continuously via diet
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
20 m / 20 f
Control animals:
yes, plain diet
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: once daily including a weekly palpation for tissue masses

BODY WEIGHT: Yes
Time schedule for examinations: weekly up to week 15, every two weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption: weekly up to week 15, every two weeks thereafter
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY: no

OPHTHALMOSCOPIC EXAMINATION: Yes
Time schedule for examinations: during acclimatization and during weeks 26 and 52
Dose groups that were examined: control and high dose group

HAEMATOLOGY: Yes
Time schedule for collection of blood: after 26 and 52 weeks
Anaesthetic used for blood collection: yes (light isoflurane anaesthesia)
Animals fasted: Yes, for 18 h with access to water
How many animals: 20 m /20 f per group
Parameters checked : Erythrocyte count, haemoglobin, haematocrit, MCV, MCH, MCHC, platelets, reticulocyte, reticulocyte fluorescence ratios, nucleated erythrocytes, total leukocytes, differential count, rec cell morphology, thromboplastin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: after 26 and 52 weeks
Anaesthetic used for blood collection: yes (light isoflurane anaesthesia)
Animals fasted: yes, for 18 h with access to water
How many animals: 20 m /20 f per group
Parameters checked : Glucose, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, asparatat aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatinine kinase, alkaline phosphatase, gamma-glutamyl-transferase, sodium, potassium, chloride, calcium, phosphorus, protein, albumin, globulin, albumin/globulin ratio

URINALYSIS: yes
Time schedule for collection of urine: after 26 and 52 weeks
Metabolism cages used for collection of urine: yes
Animals fasted: yes, 18 h
Parameters checked: Specific gravity, osmolality, colour, appearance, volume (18 h), protein, glucose, ketone, urobilinogen, bilirubin, blood, sediment

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes, all animals from control and high dose group (each 20 m /20 f) as well as all animals which died spontaneously or were killed in extremis, and all gross lesions from all animals.
Other examinations:
Organ weights were recorded from all animals on the scheduled dates of necropsy: Adrenal glands, brain, heart, kidneys, liver, spleen, testes
Statistics:
Anova (for clinical laboratory data)
Dunnett-test (many to one t-test) (for body weight, food consumption, organ weights)
Steel test (many-one rank test) (for body weight, food consumption, organ weights)
Fisher's exact test (for macroscopic findings)
Cochran Armitage test (for major non-neoplastic findings)
Peto-test (prevalence and death rate method) for tumors

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No effects

BODY WEIGHT AND WEIGHT GAIN: Mean body weight was decreased after 52 weeks in mid and high dose males by -7% and -15%, and in mid and high dose females by -11% and -8%, respectively.

FOOD CONSUMPTION AND COMPOUND INTAKE: Average food consumption during treatment for 52 weeks was slightly lower in all treated groups, but there was no clear dose-response and therefore this finding was considered not biologically relevant

CLINICAL CHEMISTRY: Effects on blood chemistry in males comprised a gradual decrease of plasma glucose in groups 3 and 4 at week 27 and in group 4 at week 53, and a lower total bilirubin level at weeks 27 and 53 in groups 2, 3 and 4. Urea was slightly increased in group 4 at week 53. A significant increase in the alanine aminotransferase level was noticed in group 4 at weeks 27 and 53. Higher albumin and lower globulin levels were evident in group 4 at week 53 or 27, leading to a higher albumin/globulin ratio in group 4 at week 27 and 53. In females, a decrease in plasma glucose in groups 4 at week 27 and 53, and a gradual decrease in total bilirubin in groups 3 and 4 at weeks 27 and 53 were present. An increase of urea concentration was observed in groups 3 and 4 at week 27 and in group 4 at week 53. A higher value of triglycerides was present in group 4 at week 27. A significant decrease in the aspartate aminotransferase level was present in group 4 at week 27. Lower globulin levels were evident in groups 3 and 4 at weeks 27 and 53, leading to a higher albumin/globulin ratio in these treatment groups.

URINALYSIS: Males of the low, mid and high dose groups excreted higher urine volumes at week 53. In addition, males of the mid and high dose had lower urinary pH-values at weeks 27 and 53 and a lower protein content in the mid and high dose at week 53.

ORGAN WEIGHTS: For males of the high dose group, a significantly higher relative liver weight was recorded (+24%). In females a significant increase in absolute mean liver weights was noted in the high dose group (+16%).

GROSS PATHOLOGY: Macroscopic findings were restricted to the liver of the high dose group and consisted of an accentuated lobular pattern in 5 males and enlargement of the liver in 5 males and 5 females.

HISTOPATHOLOGY (NON-NEOPLASTIC): After 52 weeks, a minimal centrilobular hepatocellular hypertrophy was diagnosed in 2 males and 1 female of the high dose group

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
ca. 100 - ca. 300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: NOEL based on reduction in body weigths at >= 300 mg/kg bw in males and at 1000 mg/kg bw in females
Dose descriptor:
NOAEL
Effect level:
ca. 300 - 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table: Summary of test item-related findings for ATBC, chronic part over 52 weeks

  

Intended test item intake (mg/kg bw/day)

 

Achieved intake (males)

 

Achieved intake (females)

0

 

 

 

 

 

 

100

 

 

100.16

 

 

100.86

300

 

 

302.48

 

 

304.11

1000

 

 

1003.95

 

 

1021.17

 

 

 

 

 

Body weights

Males (means-g)

 

 

 

 

Week 53

573

557

531

489**

Females (means-g)

 

 

 

 

Week 53

324

314

289*

299

Clinical chemistry

Males (means)

 

 

 

 

Glucose (mmol/L)

 

 

 

 

Week 27

 

 

Week 53

 

 

 

Bilirubin (µmol/L)

 

 

 

 

Week 27

 

Week 53

 

Urea (mmol/L)

 

 

 

 

Week 53

 

 

 

ALAT (U/L)

 

 

 

Week 27

 

 

 

Week 53

 

 

 

 

Albumin (g/L)

 

 

 

 

Week 27

 

 

 

Week 53

 

 

 

Globulin (g/L)

 

 

 

Week 27

 

 

 

Week 53

 

 

 

 

Alb/Glob ratio (rel 1)

 

 

 

 

Week 27

 

 

 

Week 53

 

 

 

 

Females (means)

 

 

 

 

Glucose (mmol/L)

 

 

 

 

Week 27

 

 

 

Week 53

 

 

 

Bilirubin (µmol/L)

 

 

 

 

Week 27

 

 

Week 53

 

 

Urea (mmol/L)

 

 

 

 

Week 27

 

 

Week 53

 

 

 

Triglyceride (mmol/L)

 

 

 

 

Week 27

 

 

 

ASAT (U/L)

 

 

 

Week 27

 

 

 

Globulin (g/L)

 

 

 

Week 27

 

 

Week 53

 

 

Alb/Glob ratio (rel 1)

 

 

 

 

Week 27

 

 

Week 53

 

 

 

 

 

 

 

Urinalysis

Males (means)

 

 

 

 

Volume (mL)

 

 

 

 

Week 53

 

pH-value

 

 

 

 

Week 27

 

 

Week 53

 

 

Protein content

 

 

 

 

Week 53

 

 

 

 

 

 

 

Organ weights

Liver (means)

 

 

 

 

Males (abs., gram)

8.83

9.30

9.58

10.86**

Males (rel., %)

2.34

2.38

2.51

2.86**

 

 

 

 

 

Females (abs., gram

5.88

6.12

5.80

7.10**

Females (rel., %)

2.65

2.71

2.76

3.03*

 

 

 

 

 

Macroscopic findings

Liver (no. affected/20)

0/10

0/10

0/10

2/10

Males: Accentuated lobular pattern

Males: Enlargement

Females: Enlargement

 

0/20

0/20

0/20

 

0/20

0/20

0/20

 

0/20

0/20

0/20

 

5/20

5/20

5/20

Histopathology

Liver (no. affected/20)

 

 

 

 

Males: Hepatocellular hypertrophy (minimal)

 

Females: Hepatocellular hypertrophy (minimal)

 

 

0/20

 

 

0/20

 

0/20

 

 

0/20

 

0/20

 

 

0/20

 

2/20

 

 

1/20

 

  • ↑ = increased
  • ↓ = reduced
  • * = 5% level (Anova and/or Dunnett-Test)
  • ** = 1% level (Anova and/or Dunnett-Test)

 

 

Applicant's summary and conclusion

Conclusions:
The results of this study indicated that the dietary administration of ATBC had no effects on survival, clinical signs, ophthalmoscopic examinations, food consumption and haematology parameters. The liver was found to be the target organ and the NOAEL in the chronic part of the study after 52 weeks was at 300 mg/kg bw/d in males and 1000 mg/kg bw/d in females based on effects to body weights at 1000 mg/kg bw/d in males and on the slight increased in liver weights and on centrilobular hypertrophy in the liver noted at 1000 mg/kg bw/da in both sexes.
Executive summary:

ATBC was administered to rats via diet in a combined chronic/carcinogenicity study over 2 years. For the chronic toxicity part, an interim sacrifice was performed after 52 weeks (results for this part are reported here).

Intended daily intake was at 100, 300 and 1000 mg/kg bw/d and the actually achieved doses were met with very low deviations from nominal.

The following results were obtained after the 52 weeks chronic period:

ATBC induced slight reductions in body weight and food consumption. Changes in clinical chemistry parameters were defined to various parameters indicating adaptive changes of metabolic activation, which were not considered to be of primary toxicological relevance. Further, the adaptive changes were expressed by few macroscopically discernible liver changes, liver weight increase and minimal hepatocellular hypertrophy in individual animals. The NOAEL was at 300 mg/kg bw/d for males and 1000 mg/kg bw/d for females. The NOEL was defined to be at 100 mg/kg bw/d for males and 300 mg/kg bw/d for the females.