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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September from 10th to 29th, 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Only four out of five strains reccomanded into the updated OECD guideline were tested
Justification for type of information:
Justification for read-across is detailed in the report attached to the IUCLID section 13.
Cross-reference
Reason / purpose:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
September from 10th to 29th, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Only four out of five strains reccomanded into the updated OECD guideline were tested
Justification for type of information:
Justification for read-across is detailed in the report attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: the bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Preculture: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 μl ampicillin (25 μg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth, 5 g NaCl.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
MAIN TEST (exp. I and II): 33, 100, 333, 1000, 2500 and 5000 μg/plate
PRE-EXPERIMENT FOR TOXICITY: 3, 10.0, 133, 100, 333, 1000, 2500 and 5000 μg/plate
Vehicle / solvent:
- Solvent: on the day of the experiment, the test article was dissolved in DMSO.
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. No precipitation of the test article occurred up to the highest investigated dose.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION
- Method: experiment I plate incorporation test; experiment II pre-incubation test.
- Incubation: the bacterial culture was incubated in a shaking water bath for 8 hours at 37 °C.
- Overlay Agar content per litre: 6.0 g MERCK Agar Agar, 6.0 g NaCl, 10.5 mg L-Histidine x HCl x H20, 12.2 mg Biotin.
- Sterilisations: performed at 121 °C in an autoclave.

REPLICATES: for each strain and dose level, including the controls three plates were used.

EXPERIMENTAL PERFORMANCE
- The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control);
500 μl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation);
100 μl Bacteria suspension (cf. test system, pre-culture of the strains);
2000 μl Overlay agar
- Pre-incubation assay: 100 μl test solution, 500 μl S9 mix / S9 mix substitution buffer and 100 μl bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes.
- Post pre-incubation: after pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
- Incubation: after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

PRE-EXPERIMENT FOR TOXICITY
- Strains: pre-experiment was performed with strains TA 98 and TA 100.
- Concentrations: 8 concentrations were tested.
- Replicate: mutatation induction with each 3 plates.
- Test conditions: the experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).
- Toxicity definition: toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- Anima source: the S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. Phénobarbital i.p. dissolved in deionised water and ß-Naphthoflavone orally dissolved in corn oil on three subsequent days.
- Liver collection: after cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The livers were prepared 24 hours after the last treatment.
- Homogenate: it was diluted 1+3 in KCl and centrifuged at 9000 g for 10 minutes at 4° C.
- Storage of stock solution: a stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to one week before use.
- Protein content: the protein content was determined using an analysis kit of Bio-Rad Laboratories. The protein concentration in the S9 preparation was 24.7 mg/ml.
- S9 mix preparation: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix: 8mM MgCl2, 33 mM KCl, 5mM Glucose-6-phosphate, 5mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. The S9 mix preparation was performed according to Ames et al.
- Storage: during the experiment the S9 mix was stored in an ice bath.

ACCEPTED CONDITIONS
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.
Range of spontaneous reversion frequencies: TA 1535 10-29; TA 1537 5 - 28; TA 98 15 - 57; TA 100 77 - 189.
Evaluation criteria:
A test article is considered positive if either a biologically relevant and reproducible dose related 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 biologically relevant and reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one 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.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment at any concentration 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. They showed a distinct increase in induced revertant colonies.
In conclusion, it can be stated that during the described 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.

Summary of revertants/plate, mean from three plates - without S9

Concentration μg/plate TA 1535 TA 1537 TA 98 TA 100
I II I II I II I II
Negative control 12 28 15 9 17 25 95 101
Solvent control 15 25 12 9 17 20 96 97
Positive control* 927 997 135 114 522 668 700 924
33 16 25 12 15 12 19 109 105
100 17 21 10 12 11 22 101 106
333 18 24 12 14 12 25 97 116
1000 16 28 9 12 12 22 96 121
2500 14 21 8 10 13 21 89 102
5000 18 16 8 11 15 30 86 107

*Sodium azide (10.0 μg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strains TA 1537 (50 μg/plate) and TA 98 (10.0 μg/plate)

Summary of revertants/plate, mean from three plates - with S9

Concentration μg/plate TA 1535 TA 1537 TA 98 TA 100
I II I II I II I II

Negative control

17 20 13 26 18 30 116 131

Solvent control*

19 23 19 21 18 28 118 110

Positive control

194 191 83 86 426 445 594 453

33

22 19 17 22 19 33 114 132

100

16 20 14 18 17 30 132 123

333

22 22 17 18 15 35 114 131

1000

15 21 14 25 19 28 118 138

2500

16 19 20 18 17 39 119 125

5000

14 21 18 20 15 33 108 133

*2-aminoanthracene (2.5 μg/plate) strains TA 1535, TA 1537, TA 98, and TA 100

Pre-Experiment for Toxicity

Substance Concentration μg/plate TA 98 TA 100
- + - +

Negative control

- 17 18 95 116

Solvent control

- 17 18 96 118

4-NOPD

10.0 522 / / /

NaN3

10.0 / / 700 /

2-AA

2.5 / 426 / 594

Test item

3 17 21 90 112
10.0 16 17 99 116
133 12 19 109 114
100 11 17 101 132
333 12 15 97 114
1000 12 19 96 118
2500 13 17 89 119
5000 15 15 86 108
Conclusions:
The test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

The study was performed to investigate the potential of test item 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, 100, 333, 1000, 2500 and 5000 μg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment 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.

Conclusion

In conclusion, 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, test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: the bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).
- Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
- Preculture: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 μl ampicillin (25 μg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth, 5 g NaCl.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
MAIN TEST (exp. I and II): 33, 100, 333, 1000, 2500 and 5000 μg/plate
PRE-EXPERIMENT FOR TOXICITY: 3, 10.0, 133, 100, 333, 1000, 2500 and 5000 μg/plate
Vehicle / solvent:
- Solvent: on the day of the experiment, the test article was dissolved in DMSO.
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. No precipitation of the test article occurred up to the highest investigated dose.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine // 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION
- Method: experiment I plate incorporation test; experiment II pre-incubation test.
- Incubation: the bacterial culture was incubated in a shaking water bath for 8 hours at 37 °C.
- Overlay Agar content per litre: 6.0 g MERCK Agar Agar, 6.0 g NaCl, 10.5 mg L-Histidine x HCl x H20, 12.2 mg Biotin.
- Sterilisations: performed at 121 °C in an autoclave.

REPLICATES: for each strain and dose level, including the controls three plates were used.

EXPERIMENTAL PERFORMANCE
- The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control);
500 μl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation);
100 μl Bacteria suspension (cf. test system, pre-culture of the strains);
2000 μl Overlay agar
- Pre-incubation assay: 100 μl test solution, 500 μl S9 mix / S9 mix substitution buffer and 100 μl bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes.
- Post pre-incubation: after pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
- Incubation: after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

PRE-EXPERIMENT FOR TOXICITY
- Strains: pre-experiment was performed with strains TA 98 and TA 100.
- Concentrations: 8 concentrations were tested.
- Replicate: mutatation induction with each 3 plates.
- Test conditions: the experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).
- Toxicity definition: toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- Anima source: the S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. Phénobarbital i.p. dissolved in deionised water and ß-Naphthoflavone orally dissolved in corn oil on three subsequent days.
- Liver collection: after cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The livers were prepared 24 hours after the last treatment.
- Homogenate: it was diluted 1+3 in KCl and centrifuged at 9000 g for 10 minutes at 4° C.
- Storage of stock solution: a stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to one week before use.
- Protein content: the protein content was determined using an analysis kit of Bio-Rad Laboratories. The protein concentration in the S9 preparation was 24.7 mg/ml.
- S9 mix preparation: before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix: 8mM MgCl2, 33 mM KCl, 5mM Glucose-6-phosphate, 5mM NADP, in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. The S9 mix preparation was performed according to Ames et al.
- Storage: during the experiment the S9 mix was stored in an ice bath.

ACCEPTED CONDITIONS
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.
Range of spontaneous reversion frequencies: TA 1535 10-29; TA 1537 5 - 28; TA 98 15 - 57; TA 100 77 - 189.
Evaluation criteria:
A test article is considered positive if either a biologically relevant and reproducible dose related 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 biologically relevant and reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one 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.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment at any concentration 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. They showed a distinct increase in induced revertant colonies.
In conclusion, it can be stated that during the described 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.

Any other information on results incl. tables

Summary of revertants/plate, mean from three plates - without S9

Concentration μg/plate TA 1535 TA 1537 TA 98 TA 100
I II I II I II I II
Negative control 12 28 15 9 17 25 95 101
Solvent control 15 25 12 9 17 20 96 97
Positive control* 927 997 135 114 522 668 700 924
33 16 25 12 15 12 19 109 105
100 17 21 10 12 11 22 101 106
333 18 24 12 14 12 25 97 116
1000 16 28 9 12 12 22 96 121
2500 14 21 8 10 13 21 89 102
5000 18 16 8 11 15 30 86 107

*Sodium azide (10.0 μg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strains TA 1537 (50 μg/plate) and TA 98 (10.0 μg/plate)

Summary of revertants/plate, mean from three plates - with S9

Concentration μg/plate TA 1535 TA 1537 TA 98 TA 100
I II I II I II I II

Negative control

17 20 13 26 18 30 116 131

Solvent control*

19 23 19 21 18 28 118 110

Positive control

194 191 83 86 426 445 594 453

33

22 19 17 22 19 33 114 132

100

16 20 14 18 17 30 132 123

333

22 22 17 18 15 35 114 131

1000

15 21 14 25 19 28 118 138

2500

16 19 20 18 17 39 119 125

5000

14 21 18 20 15 33 108 133

*2-aminoanthracene (2.5 μg/plate) strains TA 1535, TA 1537, TA 98, and TA 100

Pre-Experiment for Toxicity

Substance Concentration μg/plate TA 98 TA 100
- + - +

Negative control

- 17 18 95 116

Solvent control

- 17 18 96 118

4-NOPD

10.0 522 / / /

NaN3

10.0 / / 700 /

2-AA

2.5 / 426 / 594

Test item

3 17 21 90 112
10.0 16 17 99 116
133 12 19 109 114
100 11 17 101 132
333 12 15 97 114
1000 12 19 96 118
2500 13 17 89 119
5000 15 15 86 108

Applicant's summary and conclusion

Conclusions:
The test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

The study was performed to investigate the potential of test item 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, 100, 333, 1000, 2500 and 5000 μg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No relevant increase in revertant colony numbers of any of the four tester strains was observed following treatment 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.

Conclusion

In conclusion, 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, test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.