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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Comparable to guideline study, published in peer-reviewed literature, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Mirsalis J C and Butterworth B E (1980) Detection of unscheduled DNA synthesis in hepatocytes isolated from rats treated with genotoxic agents. An in vivo-in vitro assay for potential mutagens and carcinogens. Carcinogenesis 1, 621-625
Deviations:
no
GLP compliance:
not specified
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ZDEC
- Physical state: fine (aggregative) white powder
- Purity: at least 98%
- Impurities (identity and concentrations): ZDBC (1000 ppm) and TETD (56 ppm)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Duration of treatment / exposure:
In experiment 1, all rats were killed 2 hours after dosing.
In experiment 2, all rats were killed 16 hours after dosing.
Frequency of treatment:
Single dose
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
1 500 mg/kg bw (total dose)
No. of animals per sex per dose:
4 males/dose
Control animals:
yes
Positive control(s):
MMS (methylmethanesulfonate)
2-AAF (2-acetyl aminofluorene)

- Route of administration: oral
- Doses / concentrations: 500 mg/kg for MMS and 200 mg/kg for 2-AAF

Examinations

Tissues and cell types examined:
liver (hepatocytes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
in preliminary toxicity trials, a maximum dose of 2000 mg/kg was employed end for ZDEC, this proved highly toxic.

METHOD OF ANALYSIS:
Hepatocytes were isolated by liver perfusion and cultured in medium supplemented with tritiated thymidine. The amount of unscheduled DNA synthesis was assessed by autoradiography using standard techniques: 50 cells/slide from each of two slides/animal were evaluated. Nuclear grain count (NG), cytoplasmic grain count (CG) and net nuclear grain count (NNG: calculated as NG minus CG) were determined for each scored cell.
Statistics:
The results from ZDEC treated animals were compared statistically to those from concurrent vehicle controls using Student's t-test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
ambiguous
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Most rats dosed with ZDEC showed diarrhoea, and most killed 16 hr post-dose showed weight loss. Hepatocyte viabilities after isolation were high (69­93%), and did not differ markedly between test and control groups. A total of four treated rats killed 16 hr post-dose (three dosed at 1000 mg/kg, one each at 500 and 1500 mg/kg) showed small increases in NNG such that their individual values for this parameter exceeded 1.0 (a threshold for consideration of a positive result in the liver UDS assay). These rats also showed increased numbers of cells undergoing DNA synthesis (cells in repair: %IR) so that %IR was between 18 and 32%. However only one of these animals showed NNG greater than 0 on both of the replicate slides scored for grain count; in addition, no dose-relationship or other response pattern was evident, and (most importantly) evaluation on a group basis found no significant effect of ZDEC treatment at any dose (see statistical analysis, Table 1). It was concluded that this study had produced no conclusive evidence of induction of UDS by ZDEC: the study was considered to have shown an equivocal result.

Any other information on results incl. tables

Table 1: Rat hepatocyte UDS assay. Grain counts 2 hours and 16 hours after animal treatment:group means±standard deviation four rats/ group

ZDEC: Hepatocytes taken 2 hours post-dose

Treatment:

ZDEC (mg/kg)

Nuclear grain count

Cytoplasmic grain count

Net nuclear grain count

% cells in repair

0 (corn oil)

3.58±0.59

5.38±0.87

-1.80±0.40

1.75

500

3.15±0.35

5.04±0.48

-1.88±0.60

7.25

1000

3.49±0.48

4.96±0.94

-1.47±0.91

2.25

1500

4.16±1.09

5.70±0.74

-1.54±1.30

3.00

MMS: 500

9.13±0.85**

4.23±0.69

4.90±0.85**

40.33

ZDEC: Hepatocytes taken 16 hours post-dose

Treatment:

ZDEC (mg/kg)

Nuclear grain count

Cytoplasmic grain count

Net nuclear grain count

% cells in repair

0 (corn oil)

4.75±1.21

6.86±1.05

-2.11±1.24

5.5

500

6.73±2.09

8.22±1.55

-1.49±2.10

7.5

1000

7.65±2.13

7.26±0.85

-0.40±2.77

17.25

1500

6.60±2.06

7.94±1.55

-1.34±1.94

8

2-AAf: 200

12.77±2.11**

6.16±1.92

6.61±0.98**

55.75

MMS= positive control 1(methylmethanesulfonate).Data from three rats/group only in ZDEC study

2-AAF= positive control 2 (2-acetyl aminofluorene)

Significant difference fromvehicle controls: *test group>controls, 0.01 < P < 0,05; **test group>controls, P<0.01

Typical historicalcontrol values forthe testing laboratory were between -3 and -6.

Applicant's summary and conclusion