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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay / Ames test): negative with and without metabolic activation Gürtler & Görke 1997

Link to relevant study records
Reference
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:
from November to December 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 May 1983
Deviations:
yes
Remarks:
- TA 102 or E.coli WP2 strains are not included; only one experiment (direct plate incorporation procedure) was performed
Principles of method if other than guideline:
The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene locus
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male Wistar rat liver S9 mix
Test concentrations with justification for top dose:
100, 250, 500, 1000, 2500, 5000 µg/plate








Vehicle / solvent:
phosphate buffer ((pH 7.4, 0.1 M)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
cyclophosphamide
other: anthracen-2-amine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration triplicate
- Number of independent experiments; 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E-06 dilution
- Test substance added in medium; in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicroscopically.

Evaluation criteria:
The plates were scored for the number of mutant colonies with an automated colony counter (ArtekM 982B, Artek Systems Corporation, Farrningdale, NY, USA). ln exceptional cases, where reliable automatic counting is not possible, e.g. due to distinct precipitates of the test compound, the colonies are scored manually. The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. A positive response was considered if the number of revertants of the compound groups compared to the number of revertants of the negative control group was reproducibly higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Water solubility: no limited water solubility reported
- Precipitation and time of the determination: no precipitates

RANGE-FINDING/SCREENING STUDIES (if applicable): not performed, tested up to the highest recommended concentration

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes


Ames test:
- Signs of toxicity: No
- Individual plate counts: please refer to 'Any other information on results incl. tables'
- Mean number of revertant colonies per plate and standard deviation: please refer to 'Any other information on results incl. tables'

None of the five tester strains showed increased reversion to prototrophy in the assays at 6 concentrations tested between 0.1 and 5.0 mg/plate, either in the absence or presence of S9 mix. Generally, growth inhibition of the background lawn was not observed. There were no precipitates in the agar. Negative controls and positive controls with known mutagens produced the expected numbers of revertant colonies.


TA 1535


































































































































































































































































































































































































































Substance and dose/plate



Revertants per plate



Quotient



 



-S9



M



±SD



+S9



M



±SD



-S9



+S9



Phosphate buffer



50 µL



24



26



3



13



16



3



1.0



1.0



 



 



25



 



 



19



 



 



 



 



 



 



30



 



 



17



 



 



 



 



DMSO



50 µL



42



27



13



16



16



4



1.0



1.0



 



 



18



 



 



19



 



 



 



 



 



 



22



 



 



12



 



 



 



 



Test item



0.10 mg



24



28



4



12



12



1



1.1



0.7



 



 



32



 



 



12



 



 



 



 



 



 



29



 



 



11



 



 



 



 



Test item



0.25 mg



28



26



2



21



19



3



1.0



1.1



 



 



27



 



 



19



 



 



 



 



 



 



24



 



 



18



 



 



 



 



Test item



0.5 mg



28



25



6



17



15



4



1.0



0.9



 



 



29



 



 



18



 



 



 



 



 



 



19



 



 



11



 



 



 



 



Test item



1.0 mg



27



26



4



10



13



3



1.0



0.8



 



 



30



 



 



16



 



 



 



 



 



 



22



 



 



12



 



 



 



 



Test item



2.5 mg



25



28



3



15



16



2



1.1



1.0



 



 



31



 



 



15



 



 



 



 



 



 



29



 



 



19



 



 



 



 



Test item



5.0 mg



30



27



4



14



12



3



1.0



0.7



 



 



23



 



 



12



 



 



 



 



 



 



29



 



 



9



 



 



 



 



Anthracen-2-amine



2 µg



33



31



3



105



118



11



1.2



7.2



 



 



32



 



 



126



 



 



 



 



 



 



28



 



 



122



 



 



 



 



Cyclophosphamide



400.0 µg



49



53



13



463



451



13



2.0



27.6



 



 



42



 



 



454



 



 



 



 



 



 



67



 



 



437



 



 



 



 



Sodium azide



5.0 µg



627



622



6



201



177



21



23.6



10.8



 



 



624



 



 



165



 



 



 



 



 



 



615



 



 



164



 



 



 



 



 


TA100


































































































































































































































































































































































































































Substance and dose/plate



Revertants per plate



Quotient



 



-S9



M



±SD



+S9



M



±SD



-S9



+S9



Phosphate buffer



50 µL



110



114



3



134



121



12



1.0



1.0



 



 



116



 



 



119



 



 



 



 



 



 



115



 



 



111



 



 



 



 



DMSO



50 µL



143



120



20



92



100



8



1.1



0.8



 



 



109



 



 



99



 



 



 



 



 



 



109



 



 



108



 



 



 



 



Test item



0.1 mg



129



122



12



88



105



18



1.1



0.9



 



 



109



 



 



105



 



 



 



 



 



 



129



 



 



123



 



 



 



 



Test item



0.25mg



134



118



15



110



101



9



1.0



0.8



 



 



105



 



 



93



 



 



 



 



 



 



114



 



 



101



 



 



 



 



Test item



0.5mg



123



122



17



96



102



6



1.1



0.8



 



 



105



 



 



105



 



 



 



 



 



 



138



 



 



106



 



 



 



 



Test item



1.0 mg



117



117



6



107



101



5



1.0



0.8



 



 



111



 



 



98



 



 



 



 



 



 



123



 



 



98



 



 



 



 



Test item



2.5 mg



122



108



18



116



115



3



0.9



1.0



 



 



87



 



 



112



 



 



 



 



 



 



114



 



 



118



 



 



 



 



Test item



5.0 mg



48 B



44



3



104



102



2



0.4



0.8



 



 



43



 



 



100



 



 



 



 



 



 



42



 



 



101



 



 



 



 



Anthracen-2-amine



2.0 µg



110



111



4



662



747



82



1.0



6.2



 



 



107



 



 



755



 



 



 



 



 



 



115



 



 



825



 



 



 



 



Benzo[a]pyrene



2.5 µg



107



104



11



573



571



39



0.9



4.7



 



 



92



 



 



531



 



 



 



 



 



 



113



 



 



608



 



 



 



 



Sodium azide



5.0 µg



669



694



43



206



215



8



6.1



1.8



 



 



669



 



 



221



 



 



 



 



 



 



743



 



 



217



 



 



 



 



 


TA1537






























































































































































































































































































































































































Substance and dose/plate



Revertants per plate



Quotient



 



-S9



M



±SD



+S9



M



±SD



-S9



+S9



Phosphate buffer



50 µL



14



13



3



14



17



4



1.0



1.0



 



 



15



 



 



15



 



 



 



 



 



 



9



 



 



22



 



 



 



 



DMSO



50 µL



14



11



3



13



16



5



0.9



0.9



 



 



10



 



 



22



 



 



 



 



 



 



9



 



 



13



 



 



 



 



Test item



0.1 mg



14



10



4



10



17



7



0.8



1.0



 



 



8



 



 



16



 



 



 



 



 



 



7



 



 



24



 



 



 



 



Test item



0.25 mg



10



8



2



18



15



3



0.7



0.9



 



 



7



 



 



15



 



 



 



 



 



 



8



 



 



13



 



 



 



 



Test item



0.5 mg



11



12



4



21



19



2



0.9



1.1



 



 



9



 



 



17



 



 



 



 



 



 



16



 



 



18



 



 



 



 



Test item



1.0 mg



13



13



4



18



18



1



1.1



1.0



 



 



17



 



 



17



 



 



 



 



 



 



10



 



 



18



 



 



 



 



Test item



2.5 mg



16



17



1



18



18



2



1.3



1.1



 



 



17



 



 



17



 



 



 



 



 



 



17



 



 



20



 



 



 



 



Test item



5.0 mg



14



13



2



13



13



0



1.0



0.8



 



 



10



 



 



13



 



 



 



 



 



 



14



 



 



13



 



 



 



 



Anthracen-2-amine



2.0 µg



14



12



3



85



76



13



0.9



4.5



 



 



14



 



 



61



 



 



 



 



 



 



8



 



 



83



 



 



 



 



9-Aminoacidine



100.0 µg



1018



987



44



784



872



76



77.9



51.3



 



 



1005



 



 



917



 



 



 



 



 



 



937



 



 



914



 



 



 



 



 


TA1538






























































































































































































































































































































































































Substance and dose/plate



Revertants per plate



Quotient



 



-S9



M



±SD



+S9



M



±SD



-S9



+S9



Phosphate buffer



50 µL



12



9



3



22



22



7



1.0



1.0



 



 



6



 



 



15



 



 



 



 



 



 



8



 



 



28



 



 



 



 



DMSO



50 µL



16



13



3



23



24



1



1.5



1.1



 



 



11



 



 



23



 



 



 



 



 



 



12



 



 



25



 



 



 



 



Test item



0.1 mg



10



9



1



30



29



1



1.1



1.4



 



 



10



 



 



30



 



 



 



 



 



 



8



 



 



28



 



 



 



 



Test item



0.25 mg



12



12



2



24



25



4



1.4



1.1



 



 



14



 



 



21



 



 



 



 



 



 



10



 



 



29



 



 



 



 



Test item



0. 5 mg



12



12



2



27



25



7



1.4



1.1



 



 



10



 



 



17



 



 



 



 



 



 



14



 



 



30



 



 



 



 



Test item



1.0 mg



4



7



3



20



27



6



0.8



1.2



 



 



10



 



 



31



 



 



 



 



 



 



7



 



 



29



 



 



 



 



Test item



2.5 mg



16



12



4



36



35



2



1.4



1.6



 



 



8



 



 



33



 



 



 



 



 



 



12



 



 



35



 



 



 



 



Test item



5.0 mg



10



12



2



33



33



7



1.4



1.5



 



 



12



 



 



40



 



 



 



 



 



 



14



 



 



27



 



 



 



 



Anthracen-2-amine



2.0 µg



8



8



2



583



591



13



1.0



27.3



 



 



10



 



 



583



 



 



 



 



 



 



7



 



 



606



 



 



 



 



2-Nitrofluorene



10.0 µg



1128



1124



66



385



389



5



129.7



18.0



 



 



1188



 



 



387



 



 



 



 



 



 



1057



 



 



395



 



 



 



 



 


TA98


































































































































































































































































































































































































































Substance and dose/plate



Revertants per plate



Quotient



 



-S9



M



±SD



+S9



M



±SD



-S9



+S9



Phosphate buffer



50 µL



20



21



6



51



49



3



1.0



1.0



 



 



27



 



 



49



 



 



 



 



 



 



15



 



 



46



 



 



 



 



DMSO



50 µL



25



20



4



41



50



8



1.0



1.0



 



 



17



 



 



55



 



 



 



 



 



 



19



 



 



53



 



 



 



 



Test item



0.1 mg



14



19



6



51



48



6



0.9



1.0



 



 



25



 



 



51



 



 



 



 



 



 



17



 



 



41



 



 



 



 



Test item



0.25 mg



19



17



3



41



53



10



0.8



1.1



 



 



19



 



 



56



 



 



 



 



 



 



13



 



 



61



 



 



 



 



Test item



0.5 mg



16



21



7



45



51



7



1.0



1.0



 



 



29



 



 



49



 



 



 



 



 



 



17



 



 



59



 



 



 



 



Test item



1.0 mg



30



30



3



46



49



4



1.5



1.0



 



 



33



 



 



49



 



 



 



 



 



 



27



 



 



53



 



 



 



 



Test item



2.5 mg



19



23



10



67



60



8



1.1



1.2



 



 



16



 



 



52



 



 



 



 



 



 



34



 



 



62



 



 



 



 



Test item



5.0 mg



14



16



4



41



47



8



0.8



1.0



 



 



14



 



 



43



 



 



 



 



 



 



21



 



 



56



 



 



 



 



Anthracen-2-amine



2.0 µg



19



20



2



630



615



19



1.0



12.6



 



 



18



 



 



594



 



 



 



 



 



 



22



 



 



621



 



 



 



 



Benzo[a]pyrene



2.5 µg



21



17



5



317



323



7



0.8



6.6



 



 



18



 



 



330



 



 



 



 



 



 



12



 



 



322



 



 



 



 



2-Nitrofluorene



10.0 µg



1042



1075



33



342



401



57



52.0



8.2



 



 



1076



 



 



455



 



 



 



 



 



 



1107



 



 



407



 



 



 



 



 


M=mean


B= Background lawn reduced


± SD=Standard Deviation


+S9= With S9 mix


-S9=Without S9 mix


Quotient= Mean revertants (test substance)/Mean revertants (Solvent)


 

Conclusions:
The mutagenic potential of the test substance was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5000 µg/plate. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level in the presence and absence of metabolic activation. Therefore, the test substance was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 26 May, 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Triamine Dihydrochloride in phosphate buffer at concentrations of 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.


The test item was tested limit concentration 5000 µg/plate All of the five tester strains showed no increased reversion to prototrophy at all concentrations tested, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was observed only at the highest concentration tested (5000 µg/plate) in the TA100 strain without metabolic activation. There were no precipitates in the agar found at any concentration in all strains used in the tests without and with S9 mix.


This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.


Evaluation of the data does indicate that the test substance is not a mutagen in the Ames-Test. In the five tester strains (all strains of Salmonella typhimurium) no increased reversion to prototrophy with the test substance at the doses tested in the absence and presence of S9 mix were observed.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 26 May, 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Triamine Dihydrochloride in phosphate buffer at concentrations of 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.


The test item was tested limit concentration 5000 µg/plate. All of the five tester strains showed no increased reversion to prototrophy at all concentrations tested, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was observed only at the highest concentration tested (5000 µg/plate) in the TA100 strain without metabolic activation. There were no precipitates in the agar found at any concentration in all strains used in the tests without and with S9 mix.


This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.


Evaluation of the data does indicate that the test substance is not a mutagen in the Ames-Test. In the five tester strains (all strains of Salmonella typhimurium) no increased reversion to prototrophy with the test substance at the doses tested in the absence and presence of S9 mix were observed.


 


In a QSAR prediction using DEREK Nexus v6.1 the potential of Triamine Dihydrochloride to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have beenshown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.


The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Furthermore, the query structure does  not contain an unclassified feature and is consequently not predicted to be indeterminate in the bacterial in vitro (Ames) mutagenicity test. However, experimental data are available clearly reporting a negative result.


Based on these results Triamine Dihydrochloride is considered non-mutagenic as predicted by DEREK Nexus.


This study is classified as acceptable for assessment based on methodolgy and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

Justification for classification or non-classification

Based on the study and the QSAR prediction results a classification according to Regulation (EC) No. 1272/2008 (CLP), Annex I, is not warranted.