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Toxicological information

Specific investigations: other studies

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Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented, meets scientific principle/standards.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1989

Materials and methods

Principles of method if other than guideline:
The study was performed to determine the hepatocarcinogenic potential of test substance in rats previously treated with N-nitrosodiethylamine (DEN, initiator). After treatment with test substance/positive control, animals were sacrificed and liver samples were sectioned. The sections of liver were stained and examined to determine number/cm2 and area of γ-GT (γ-glutamyl transpeptidase) positive foci.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
carcinogenicity

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: 2,2'-[4-Aminophenyl)Imino]-Bisethanol Sulfate (A050)
- Substance type: Pure active substance
No other information on details of the test material was provided in the study report.

Test animals

Species:
rat
Strain:
other: F344/DuCrj
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: From Charles River Japan, Inc., Kanagawa
- Age at study initiation: 6 week old
- Weight at study initiation: Not reported
- Housing: Animals were housed five to a plastic cage with hardwood chips for bedding.
- Diet: Animals were provided with feed containing test substance. However, no other details on diet were provided in the study report.
- Water: Not reported
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2°C
- Humidity: 60±20%
- Air changes: 15 air changes/h
- Photoperiod: 12 h light/dark cycle

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION: No details on preparation of diet were provided in the study report.

TREATMENT: The treatment schedule was as follows:
-Group 1: Animals were treated once with N-nitrosodiethylamine (DEN) at a dose of 200 mg/kg bw intraperitoneally as an initiator. Two weeks later, all animals were divided into 4 subgroups and treated for 6 weeks with following doses:

Group 1-I: 25 animals were treated with diet containing 1000 ppm of test substance (76 mg/kg bw/d)
Group 1-II: 25 animals were treated with diet containing 330 ppm of test substance (24 mg/kg bw/d)
Group 1-III: 25 animals were treated with diet containing 110 ppm of test substance (9 mg/kg bw/d)
Group 1-IV: 25 animals were treated with diet containing 600 ppm of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) (41 mg/kg bw/d)

-Group 2: Animals were treated once with N-nitrosodiethylamine (DEN) at a dose of 200 mg/kg bw intraperitonially. Animals were not treated further.

-Group 3: Animals were treated with saline (intraperitoneally) initially. Two weeks later, all animals were divided into 4 subgroups and treated for 6 weeks with following doses:
Group 3-I: 25 animals were treated with diet containing 1000 ppm of test substance (76 mg/kg bw/d)
Group 3-II: 25 animals were treated with diet containing 330 ppm of test substance (24 mg/kg bw/d)
Group 3-III: 25 animals were treated with diet containing 110 ppm of test substance (9 mg/kg bw/d)
Group 3-IV: 25 animals were treated with diet containing 600 ppm of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) (41 mg/kg bw/d)
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
110, 330 and 1000 ppm (9, 24 and 76 mg/kg bw/d, respectively)
Basis:
nominal in diet
No. of animals per sex per dose:
Group 1: 100 animals were divided equally into 4 subgroups (25 animals each)
Group 2: 50 animals
Group 3: 100 animals were divided equally into 4 subgroups (25 animals each)
Control animals:
other: Animals treated with N-nitrosodiethylamine alone (Group 2) (200 mg/kg bw, intraperitoneally)
Details on study design:
-Three weeks after the beginning of the experiment, two-thirds partial hepatectomy was performed on all animals (Hasegawa et al., 1986; Higgins and Anderson, 1931).

SACRIFICE: All survivors were sacrificed under ether anesthesia for examination at week 8.

NECROPSY: All survivors were autopsied, the liver and kidney weights measured and the organ weight to body weight ratios calculated.

HISTOPATHOLOGICAL EXAMINATION AND QUANTITATION OF γ -GT POSITIVE FOCI: The livers were cut into 2-3 mm thick sections with a razor blade and fixed in ice-cold acetone for subsequent histochemical staining of γ -GT (Rutenberg et al., 1969; Tsuda et al., 1984) and routine hematoxylin and eosin staining. The numbers and areas of γ -GT positive foci were measured using a color video image processor (VIP-21C, Olympus-Ikegami Tsushin).

EVALUATION OF RESULTS: The results were assessed by comparing the values of γ –GT positive foci between Group 1 (DEN + test substance) and Group 2 (DEN alone). Group 3 served for the estimation of carcinogenic potential.

STORAGE OF TISSUE: Other liver sections and the kidneys were fixed in 10% buffered formalin solution and preserved.

Examinations

Examinations:
- During study, animals were observed for mortality, body weights, food consumption and test substance intake.
- During necropsy, animals were observed for gross pathological changes and organ weights were determined.
Positive control:
3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB)

Results and discussion

Details on results:
- MORTALITY: Although several animals died after the surgical operation, no treatment-related deaths occurred. Final survival rates in each group were 92% or above.
- BODY WEIGHTS: Marked growth retardation was evident in animals which received 3'-Me-DAB (Groups 1-IV and 3- IV), but not in animals exposed to test substance at any of the dietary levels (Groups 1 (I to III)) when compared to animals given DEN alone (Group 2).
- FOOD CONSUMPTION: Animals in Group 1-IV consumed less food than Group 2, but no adverse effects regarding food consumption data were found in Groups 1-I to III.
- CHEMICAL INTAKE: For the four treated subgroups (Group 1-(I-IV)), the intake of test substance or positive control (3'-Me-DAB), calculated from nominal dietary levels, the mean food consumption and the mean body weight for each subgroup, were 76, 24, 9 and 41 mg/kg bw/d, respectively.
- GROSS FINDINGS: Enlargement and discolored area or nodules of liver were observed in animals exposed to positive control (3'-Me-DAB) (Group 1-IV and 3-IV). However, no treatment-related gross liver lesions were observed in animals receiving test substance.
- ORGAN WEIGHTS: Statistically significant increase was seen in the liver weights of animals treated with positive control (3'-Me-DAB (Group 1-IV)) when compared to Group 2. The elevated relative kidney weight observed in Group 1-IV seemed to be related to growth retardation. The reduced relative kidney weight found in Group 1-II did not seem to be due to test substance treatment, since no dose-dependency was apparent.
- HISTOCHEMICAL ANALYSIS:
- The number and area of γ -GT positive foci/ cm2 in animals exposed to test substance (Group 1-I to III) were not significantly different from the values for animals treated with DEN alone (Group 2).
- The number and area of γ -GT positive foci/ cm2 in animals treated with positive control (Group 1-IV) (3'-Me-DAB) was markedly increased as compared to Group 2.
- Animals of Group 3-IV, subjected to the treatment with positive control (3'-Me-DAB) after saline injection, demonstrated marked development of γ -GT positive foci.
- In contrast, with exception of only a few γ -GT positive foci of very small size in animals of group 3-I, treated with test substance at 1,000 ppm without initiation, no lesions were observed in the other groups without DEN initiation.

Any other information on results incl. tables

Table 1: Numbers and area of γ -GT positive foci in the livers of animals initiated with N-nitrosodiethylamine (DEN) and subsequently treated with test substance/positive control (Study # 26A050)

Group

Sub-group

γ-GT positive foci

Number/cm2

Area (mm2/cm2)

1

I

8.42±4.32a

0.56±0.23

II

10.08±5.58

0.58±0.26

III

9.30±5.59

0.54±0.26

IV

67.34±21.45**

26.09±7.53**

2

8.73±5.16

0.54±0.25

3

I

0.01±0.06

0±0

II

0±0

0±0

III

0±0

0±0

IV

23.00±9.81

3.07±0.24

a= Values are means ± S.D. **= Significantly different from Group 2 at P<0.01

Applicant's summary and conclusion

Conclusions:
2,2'-[4-Aminophenyl)Imino]-Bisethanol Sulfate (A050) when administered to the male F344/DuCrj rats (previously treated with N-nitrosodiethylamine (DEN)) for 6 weeks at dose levels of 110, 330 and 1000 ppm in diets (76, 24 and 9 mg/kg bw/d, respectively), revealed that A050 was neither a hepatocarcinogen nor hepatopromoter.
Executive summary:

The study was performed to determine the hepatocarcinogenic potential of 2,2'-[4-Aminophenyl)Imino]-Bisethanol Sulfate (A050) in male rats pre-treated with N-nitrosodiethylamine.

Male rats (6 weeks old) obtained from Charles River Japan, Inc., Kanagawa were used in the study. Animals were housed five to a plastic cage with hardwood chips for bedding. During study, animals were housed in standard laboratory environmental conditions (Temperature: 22±2°C; Humidity: 60±20%; Air changes: 15 air changes/h; Photoperiod: 12 h light/dark cycle). Study involved 3 treatment groups and 2/3 treatment groups were divided into 4 subgroups. The treatment schedule was as follows:

Group 1: Animals were treated once with N-nitrosodiethylamine (DEN) at a dose of 200 mg/kg bw intraperitoneally as an initiator. Two weeks later, all animals were divided into 4 subgroups and treated for 6 weeks with following doses:

Group 1-I: 25 animals were treated with diet containing 1000 ppm of test substance (76 mg/kg bw/d)

Group 1-II: 25 animals were treated with diet containing 330 ppm of test substance (24 mg/kg bw/d)

Group 1-III: 25 animals were treated with diet containing 110 ppm of test substance (9 mg/kg bw/d)

Group 1-IV: 25 animals were treated with diet containing 600 ppm of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) (41 mg/kg bw/d)

 

Group 2: Animals were treated once with N-nitrosodiethylamine (DEN) at a dose of 200 mg/kg bw intraperitonially. Animals were not treated further.

Group 3: Animals were treated with saline (intraperitoneally) initially. Two weeks later, all animals were divided into 4 subgroups and treated for 6 weeks with following doses:

Group 3-I: 25 animals were treated with diet containing 1000 ppm of test substance (76 mg/kg bw/d)

Group 3-II: 25 animals were treated with diet containing 330 ppm of test substance (24 mg/kg bw/d)

Group 3-III: 25 animals were treated with diet containing 110 ppm of test substance (9 mg/kg bw/d)

Group 3-IV: 25 animals were treated with diet containing 600 ppm of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) (41 mg/kg bw/d)

 

Three weeks after the beginning of the experiment, two-thirds partial hepatectomy was performed on all animals. All survivors were sacrificed under ether anesthesia for examination at week 8. All survivors were autopsied, the liver and kidney weights measured and the organ weight to body weight ratios calculate.

During study, animals were observed for mortality, body weights, food consumption and test substance intake. During necropsy, animals were observed for gross pathological changes. After sacrifice, organs were collected and weights were determined.

Liver samples were sectioned and fixed (in ice-cold acetone) for subsequent histochemical staining of γ -GT (γ-glutamyl transpeptidase) and routine hematoxylin and eosin staining.

The results were assessed by comparing the values of γ –GT positive foci between Group 1 (DEN + test substance) and Group 2 (DEN alone). Group 3 served for the estimation of carcinogenic potential.

No test substance treatment related mortality was observed. No test substance treatment related changes were observed for body weight, food consumption, organ weights and histological sections of liver.

The number and area of γ -GT positive foci/ cm2 in animals exposed to test substance (Group 1-I to III) were not significantly different from the values for animals treated with DEN alone (Group 2).

The number and area of γ -GT positive foci/ cm2 in animals treated with positive control (3'-Me-DAB) were markedly increased as compared to Group 2. Animals of Group 3-IV, subjected to the treatment with positive control (3'-Me-DAB) after saline injection, demonstrated marked development of γ -GT positive foci.

Based on above, 2,2'-[4-Aminophenyl)Imino] Bisethanol Sulfate (A050) when administered to the male F344/DuCrj rats (previously treated with N-nitrosodiethylamine) for 6 weeks in diets at dose levels of 110, 330 and 1000 ppm revealed that A050 was neither a hepatocarcinogen nor a hepatopromoter.