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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
UKEMS guidelines
Deviations:
yes
Remarks:
only large mutant colonies (diameter > 0.5 mm) were scored
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

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)
- Lot/batch No.: 2036 92001

Method

Target gene:
Thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
other: cells heterozygous at the thymidine kinase gene locus (TK +/-)
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
0.0025, 0.005, 0.01, 0.025 and 0.05 µg/ml without metabolic activation.
0.025, 0.05, 0.1, 0.2, 0.4 and 0.6 µg/ml with metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
other: ZDMC
Details on test system and experimental conditions:
METHOD OF APPLICATION: soft agar cloning technique

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
ZDEC proved highly cytotoxic: following preliminary toxicity tests, L5178Y cells were treated at five concentrations between 0.0025 and 0.05 µg/ml in the absence of S-9 mix and 0.025-0.6 µg/ml in its presence.

Any other information on results incl. tables

In the first mutation experiment, total growth (% of control value: calculated as % suspension growth x % cloning growth /100) in test cultures ranged from 113 to 4.4% without S-9 mix and 119 to 59% in its presence. In the second experiment, these ranges were respectively 102-8% and 103-9%. Even after exposure at clearly cytotoxic concentrations of ZDEC, there were no significant increases in either mutant colony numbers or calculated mutant frequencies. It was concluded that ZDEC gave negative results in this assay. These results are summarized in Table 1.

In reference control cultures treated with ZDMC, there was evident cytotoxicity: total growth was 7% or 2% of control values at 0.5 µg/ml without S-9 mix, 15% of control at 1 µg/ml with S-9 mix (Experiment 2 only: in Experiment 1, ZDMC was tested at 0.7 µg/ml and total growth was reduced to only 72%). No meaningful increases in mutant colony numbers or mutation frequencies were seen in non-activated ZDMC test cultures, but with S-9 mix an apparent effect was seen in Experiment 1 only: after exposure at 0.7 µg/ml, mutant colony numbers showed a consistent (> twofold) increase over solvent controls, leading to a doubling of the calculated mutation frequency. Exposure to ZDMC at the slightly higher (and more cytotoxic) concentration of 1 µg/ml in Experiment 2 (again with S-9 mix) showed a doubling of mutant colony numbers and 1.5-fold increase in calculated mutation frequency in one of the two replicate cultures, while the duplicate culture showed no such increases. Owing to the known variation of spontaneous mutation frequency in the L5178Y TK + /- assay system, a larger (e.g. at least threefold) and clearly reproducible increase in mutation frequency is required before a positive result is concluded. It was therefore concluded from these test data that ZDMC showed no evidence of mutagenic activity without S-9 mix, and also gave a negative (or, at most, equivocal) result in the presence of the S-9 mix activating system.

Table 1: Mean total growth (TG), mutantcolony numbers(MC), mutation frequencies (MF) andinduced mutation frequencies(IMF) recorded in L5178Y TK + /- mutation assays with ZDEC

 

 

Experiment 1

Experiment 2

Treatment (µg/ml)

S-9 mix

TG

(%)

MC

(/plate)

MF

(x10-5)

IMF

(x10-5)

TG

(%)

MC

(/plate)

MF

(x10-5)

IMF

(x10-5)

DMSO (0)

-

100

62

9.1

0

100

23

4.4

0

ZDEC, 0.0025

-

113

63

9.8

+0.7

80

21

5.0

+0.6

ZDEC, 0.005

-

69

42

7.4

-1.7

99

16

3.9

-0.5

ZDEC, 0.01

-

6

42

10.9

+1.8

102

28

4.4

0

ZDEC, 0.025

-

3

36

9.4

+0.3

25

39

6.9

+2.5

ZDEC, 0.05

-

4

45

10

+0.9

8

31

7.2

+2.8

EMS*, 500

-

35

236

51.2

+42.1

35

129

52.1

+47.7

ZDMC, 0.5

-

7

42

10.2

+1.1

2

21

7.1

+3.0

DMSO (0)

+

100

42

7.0

0

100

24

4.4

0

ZDEC, 0.025

+

99

65

8.8

+1.8

NT

30

NT

NT

ZDEC, 0.05

+

111

87

12.0

+5.0

103

39

4.5

+0.1

ZDEC, 0.1

+

119

89

11.6

+4.6

88

55

6.5

+2.1

ZDEC, 0.2

+

94

42

7.3

+0.3

53

48

8.0

+3.6

ZDEC, 0.4

+

59

51

7.6

+0.6

46

32

8.9

+4.5

ZDEC, 0.6

+

NT

NT

NT

NT

9

51

8.2

+3.8

DMBA*, 5

+

7

97

31.7

+24.7

3

44

24.3

+19.9

DMBA*, 5

-

119

108

14.4

+7.4

105

44

5.7

+1.3

ZDMC, 0.7

+

72

107

15.1

+8.1

NT

NT

NT

NT

ZDMC, 1.0

+

NT

NT

NT

NT

15

31

5.1

+0.7

NT = not tested

All values are rounded to the nearest significant figure shown.

*Positive controls:EMS= ethyl methanesulfonate; DMBA = 7,12-dimethylbenzanthracene

Applicant's summary and conclusion