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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:
12 October - 22 November 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
The standard plate incorporation and the preincubation methods
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
AHTN

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9 mix
Test concentrations with justification for top dose:
Dose range 8-5000 µg/plate.
Vehicle / solvent:
Dimethyl Sulfoxide (Merck)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA1535 and TA100
Positive control substance:
sodium azide
Remarks:
SERVA, Lot 30175 - Dosage: 1.0µg/plate

Migrated to IUCLID6: without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA1537 and TA97
Positive control substance:
other: ICR 191 without metabolic activation S9
Remarks:
SERVA, Control K8 - Dosage: 1.0µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA98
Positive control substance:
2-nitrofluorene
Remarks:
ALDRICH, Lot 3734435 - Dosage: 0.5µg/plate

Migrated to IUCLID6: without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA102
Positive control substance:
mitomycin C
Remarks:
SIGMA, Lot 32 H0326 - Dosage: 0.4µg/plate

Migrated to IUCLID6: without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for E. coli WP2 uvr A
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4-Nitroquinolineoxide (SIGMA, Lot 84F-0572)

Migrated to IUCLID6: without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for all strains to examine the activity of the S9 mix
Positive control substance:
other: 2-Aminoanthracene - with and without metabolic activation S9
Remarks:
SIGMA, Lot 121 H3475
Details on test system and experimental conditions:
The Salmonella typhimurium strains TA1535, TA1537, TA97, TA98, TA100 and TA102 were obtained from B.N. Ames. E. coli WP2 uvrA was obtained from The National Collection of Industrial and Marine Bacteria Ltd, Aberdeen Scotland.
Evaluation criteria:
There was no generally accepted statistical treatment of Ames test and E.coli reversion system data. In most tests the results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA1535, TA1537, TA98 and E. coli VP2 uvrA. For strains TA97, TA100 and TA1O2 a 1.5 - fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation and biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of bis+ or trp+ revertant colonies.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
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

The results of the standard plate incorporation test are summarized in table 1 and of the preincubation test in table 2.

The spontaneous mutation rate was within the range observed for historical controls. All positive controls gave acceptable responses.

Toxic effects were not apparent in the standard assay, thus a dose range of 8 to 5000 µg/plate was evaluated to include solutions and suspensions.

AHTN did not cause en increase of the number of revertant colonies in any of the seven tester strains.

Table I Salmonella mutagenicity test (standard assay). Mean values and standard deviations.

Experiment No 01 01 01 01 01 01 02 02 02 02 02 02 02 02
Activation -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Strain TA1535 TA1535 TA1537 TA1537 WP2uvrA WP2uvrA TA97 TA97 TA9B TA98 TA100 TA100 TA102 TA102
Concentration
µg/plate
                           
0 13 9 10 10 24 22 245 245 26 28 196 197 312 298
  ±5 ±3 ±4 ±4 ±8 ±6 ±20 ±3 ±6 ± 4 ±21 ±25 ±24 ±7
8 13 7 7 11 23 20 258 257 24 32 197 207 324 292
  ±2 ±3 ±1 ±4 ±5 ±1 ±20 ±11 ±4 ± 7 ±12 ±16 ±20 ±19
40 15 12 9 11 19 26 250 251 27 27 179 208 330 314
  ±1 ±4 ±2 ±5 ±6 ±2 ±16 ±20 ± 4 ± 5 ±15 ± 7 ±18 ± 9
200 13 10 9 9 19 21 252 273 27 36 194 203 312 346
  ±5 ±3 ±1 ±2 ±5 ±5 ±17 ±25 ±4 ±11 ±14 ±17 ±27 ±17
1000 16 8 8 9 15 21 240 231 25 28 197 176 307 291
  ±3 ±2 ±1 ±2 ±2 ±6 ±19 ±30 ± 5 ± 5 ±14 ±24 ±16 ±19
5000 14 6 6 4 21 23 237 234 21 26 203 199 278 319
  ±4 ±4 ±1 ±2 ±4 ±5 ±23 ±20 ± 4 ± 2 ± 8 ± 8 ±13 ±30

Table 2 Salmonella mutagenicity test (Liquid preincubation assay). Mean values and standard deviations.

Experiment No 01 01 01 01 01 01 02 02 02 02 02 02 02 02
Activation -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Strain TA1535 TA1535 TA1537 TA1537 WP2uvrA WP2uvrA TA97 TA97 TA9B TA98 TA100 TA100 TA102 TA102
Concentration
µg/plate
                           
0 7 8 13 7 27 28 258 265 25 33 162 132 359 398
  ±4 ±2 ±2 ±1 ±8 ±6 ±14 ±10 ± 8 ±10 ±11 ±14 ± 26 ± 31
8 13 7 13 8 28 29 -287 281 24 31 110 125 358 434
  ±2 ±1 ±4 ±2 ±5 ±3 ±15 ±5 ± 6 ± 6 ±13 ± 20 ± 18 ± 22
40 11 4 11 6 35 31 285 276 21 32 98 144 342 423
  ±1 ±1 ±3 ±2 ±9 ±3 ±15 ± 3 ± 4 ±9 ±12 ±21 ± 11 ± 14
200 13 8 10 8 30 32 273 295 27 35 102 107 325 343
  ±3 ±1 ±1 ±5 ±4 ±3 ±28 ±13 ± 2 ± 3 ± 8 ±8 ± 20 ± 17
1000 11 8 9 8 26 35 298 301 23 30 100 105 217 233
  ±2 ±6 ±4 ±3 ± 6 ± 3 ±21 ±27 ± 4 ± 4 ± 7 ± 15 ± 17 ± 26
5000 12 7 11 10 33 34 304 257 27 49 107 113 256 189
  ±3 ±3 ± 4 ± 5 ±11 ± 6 ±29 ±36 ± 1 ± 14 ±10 ± 10 ± 38 ± 19

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It can be concluded that neither AHTN itself nor any metabolite generated by the metabolic activation system is mutagenic in the Ames test under the described experimental conditions.
Executive summary:

AHTN was investigated for mutagenic potential using two versions of the Ames test: the standard plate incorporation and the preincubation methods. Seven tester strains were employed (S. typhimurium TA1535, TA1537, TA97, TA98, TA100, TA1O2 and E. coli VP2 uvrA). The cells were exposed to the test compound in absence and presence of a metabolic activation systent (S9 mix) prepared from the livers of phenobarbital/8-naphthoflavone treated rats.

Responsiveness of the strains and activity of the S9 mix were verified by including appropriate positive control compounds.

The test compound was dissolved in DMSO. Milky suspensions concentrations of 158 pg/plate and above. The dose range was chosen 5000 µg/plate. No toxicity was observed up to 5000 µg/plate.

No increase of the number of reverting colonies were observed for any of the seven tester stains.

Thus it can be concluded that neither AHTN nor any of its metabolites are mutagenic in the Ames test under the described conditions.