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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 24, 1997 through March 21, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
425-950-7
EC Name:
-
Cas Number:
187393-00-6
Molecular formula:
C38-H49-N3-O5
IUPAC Name:
5-[(2-ethylhexyl)oxy]-2-(4-{4-[(2-ethylhexyl)oxy]-2-hydroxyphenyl}-6-(4-methoxyphenyl)-1,3,5-triazin-2-yl)phenol
Details on test material:
- Name of test material (as cited in study report): CGF-C-1607
- Physical state: yellow solid
- Analytical purity: > 98%
- Lot/batch No.: 003
- Expiration date of the lot/batch: August 31, 1998
- Storage condition of test material: room temperature

Method

Target gene:
his
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from livers of rats exposed to phenobarbital and B-naphthoflavone
Test concentrations with justification for top dose:
33.3, 100, 333.3, 1000, 2500, and 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: its solubility properties and its relative nontoxicity to the bacteria
Controls
Untreated negative controls:
yes
Remarks:
untreated plates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (SA); 4-nitro-o-phenylene-diamine (NOPD); 2-aminoanthracene (2-AA)
Remarks:
SA = TA 1535 and TA 100 w/out S9; NOPD = TA 1537 and TA 98 without S9; 2-AA = strains with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation in experiment I and pre-incubation in experiment II

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 8 hours




Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a biologically relevant and reproducible positive response at anyone of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.
Statistics:
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben). The counter was connected to an mM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
except pre-incubated conditions
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Results of pre-experiment for toxicity

Substance

Concentration (ug) per plate

Revertants per plate

TA98

TA 100

-

+

-

+

Negative control

-

24

35

124

127

Solvent control

-

19

29

125

118

4-NOPD

10.0

469

/

/

/

Sodium azide

10.0

/

/

972

301

2-aminoanthracene

2.5

/

944

/

301

Test article

3.3

19

27

112

115

10.0

17

24

120

133

33.3

20

28

110

125

100.0

19

28

107

130

333.3

20

27

117

142

1000.0

19

15

116

144

2500.0

20

18

150

153

5000.0

12

14

122

163

- = without S9 mix

+ = with S9 mix

/ = not performed

Table 2. Summary of results without S9 mix (Experiments I and II)

Concentration ug/plate

Revertants/plate mean from three plates

TA 1535

TA 1537

TA 98

TA 100

I

II

I

II

I

II

I

II

Negative control

11

15

8

8

24

16

124

136

Solvent control

11

17

11

7

19

20

125

131

Positive control#

529

1378

166

140

469

432

972

1329

33.3

8

16

8

11

20

18

110

124

100.0

13

15

9

8

19

16

107

135

333.3

11

15

10

8

20

14

117

142

1000.0

13

17

8

7

19

14

116

120

2500.0

7

14

6

6

20

17

150

158

5000.0

7

17

5

7

12

15

122

127

# Sodium azide (10.0 ug/plate) strains TA 1535 and TA 100

4-nitro-o-phenylene-diamine strains TA 1537 (50 ug/plate) and TA 98 (10.0 ug/plate)

Table 3. Summary of results with S9 mix (Experiments I and II)

Concentration ug/plate

Revertants/plate mean from three plates

TA 1535

TA 1537

TA 98

TA 100

I

II

I

II

I

II

I

II

Negative control

13

19

13

15

35

37

127

160

Solvent control

13

17

14

10

29

30

118

150

Positive control##

143

127

146

104

944

315

301

569

33.3

11

15

9

11

28

26

125

139

100.0

10

12

15

12

28

20

13

143

333.3

9

19

13

14

27

26

142

148

1000.0

12

18

9

15

15

20

144

146

2500.0

9

21

5

14

18

29

153

162

5000.0

8

18

7

10

14

25

163

170

## 2-aminoanthracene (2.5 ug/plate) strains TA 1535, TA 1537, TA 98, and TA100

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative all strains with and without S9

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, CGF -C-1607 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of CGF-C-1607 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 ug/plate. In experiment I, toxic effects, indicated by a reduction in the number of revertants, were observed in strain TA 1537 at 5000.0 ug/plate without S9 mix and at 2500.0 and 5000.0 ug/plate with S9 mix and in strain TA 98 at 1000.0 and 5000.0 ug/plate with S9 mix. The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with CGF -C-1607 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.