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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:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed in recognised procedure. GLP status not specified in the study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Test method conformed to the procedure published by Ames et al. Mut Res 31:347-364, 1975
Deviations:
not specified
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
None specified

Method

Target gene:
None specified
Species / strain
Species / strain / cell type:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
39, 78, 156, 312, 625 μg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide and ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: nitroflourene; 2-aminoanthracene
Details on test system and experimental conditions:
Induction of Rat Liver Enzymes for Activation
Preparation of animals:
-male rats – 200-300 gm each (Charles River, Wistar strain, Charles River Breeding Lab., Inc.)
-single injection of Aroclor 1254 (diluted in corn oil to a conc. of 200 mg/ml) I.P. at a rate of 500 mg/kg five days before sacrifice.
-Drinking water given ad libitum & Purina Laboratory Chow until 12 hours before sacrifice.
-On fifth day of induction, rats were killed by cervical separation decapitated and bled.
Preparation of liver homogenate fraction (S-9):
-All steps are at 0-4°C using cold, sterile solutions and glassware.
-Liver placed in beakers containing 0.15M KCl (approx. 1 ml/gm of wet liver) for weighing.
-After weighing, livers are transferred to a beaker containing 0.15 M KCl (3 ml/gm wet liver) minced with sterile scissors, and homogenized in a Potter-Elvejham apparatus with a Teflon pestle.
-Homogenate centrifuged for 10 minutes at 9000 x g (8700 rpm).
-Supernatant decanted and saved (S-9 fraction).
-Fresh S-9 fractions are distributed in 2 ml portions.
-Quickly frozen and stored at -80°C.
Preparation of S-9 Mix:
S-9 Mix contains per ml:
S-9 (0.04-0.1 ml) used 50 μl/ml
MGCl2 8μM
KCl 33 μM
Glucose-6-phosphate 5μM
NADP 4Μm
Sodium phosphate Buffer pH 7.4 100μM
S-9 Mix is freshly prepared each day, filter sterilized, and kept on ice before and during use.

Mutagenesis Assays on Plates
Media:
Top Agar: 0.6% Difco Agar; 0.5% NaCl
Autoclaved in 100 ml volumes and kept at room temperature before use.
Agar melted in steam and 10 ml of a sterile solution of 0.5 μM 1-Histidine HCl-0.5 μM biotin is added to the molten top agar and mixed thoroughly by gentle swirling.
Minimal-glucose agar medium in Vogel-Bonner Medium E: 1.5% Difco Agar; 2.0% Glucose

Mutagenesis Assay Procedure with S-9 Mix
Added in order to 2ml molten top agar at 45°C
-0.1 ml of overnight nutrient broth culture of bacterial tester strain
-Sample to be tested - 10μl
-0.5 ml of the S-9 Mix.
-S-9 Mix should not be left at 45°C for more than a few seconds.
The contents are mixed by rotating the tube between the palms.
The contents of the tube are then poured on minimal glucose agar plates.
Uniform distribution of the top agar is accomplished by gently tilting and rotating the uncovered plate, and then setting down to harden.
Mixing, pouring and distributing should take less than 20 seconds.

Mutagenic Assay Without Mammalian Microsomes
Same as above.
Evaluation criteria:
None specified
Statistics:
None specified

Results and discussion

Test results
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Additional information on results:
Inspection of the data reveals no evidence of mutagenicity in any of the strains at any concentration of the test chemical.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In Vitro Assay of Uniroyal Compound TVIC-B-5909 Using Salmonella typhimurium (Averages)

Compound

Metabolic Activation

μg of Compound Added per Plate

Histidine-Positive Revertants per Plate

TA-98

TA-100

TA-1535

TA-1537

TA-1538

Negative Control

-

+

 

16

33

142

191

8

11

6

5

--

20

TVIC-B-5909

-

-

-

-

-

+

+

+

+

+

39

78

156

312

625

39

78

156

312

625

13

15

17

12

15

30

29

28

32

21

116

121

128

136

149

153

139

158

138

175

4

8

9

6

10

8

10

6

8

8

2

3

4

6

2

4

5

4

5

6

--

--

--

--

--

15

20

16

14

16

MMNG

-

-

10

5

--

--

751

740

659

640

11

14

--

--

9-Aminoacridine

-

-

20

10

--

--

--

--

--

--

25

7

--

--

Nitroflourene

-

-

20

10

412

351

--

--

--

--

--

--

--

--

2-Aminoanthracene

+

+

15

5

--

556

--

373

16

--

13

--

135

--

DMSO

-

+

 

21

29

158

171

9

10

5

7

--

18

Ethanol

-

 

--

--

--

7

--

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The compound assayed was found to be non-mutagenic.
Executive summary:

The purpose of the study is to determine whether the compound (TVIC) elicited a mutagenic response in microorganism. An Aroclor 1254-stimulated, rat-liver-homogenate metabolic activation system was included in the assay procedure to provide metabolic steps that the bacteria are either incapable of conducting or that they do not carry our under the assay conditions.

The test method conformed to the procedure published by Ames et al. Mut Res 31:347-364, 1975.

Compound TVIC was examined for mutagenic activity with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538. Each assay was performed in the presence and in the absence of a metabolic activation system. The compound assayed was found to be non-mutagenic.