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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tris(2-hydroxyethyl)methylammonium methyl sulphate- Substance type: organic

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (phenobarbital and beta-naphthoflavone induced rat liver)
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2500, 5000 µg/plate with and without S9 mix
Vehicle / solvent:
- Vehicle used: water- Justification for choice of vehicle: Due to the good solubility of the test substance in ultrapure water, ultrapure water was used.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2-AA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9 mix (TA1535, TA100, TA1537, TA98, WP2 uvr A)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
MNNG
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
without S9 mix (TA1535, TA100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
NOPD
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
without S9 mix (TA98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
AAC
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
4-NQO
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix (WP2 uvr A)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubationDURATION- Preincubation period: 20 min- Exposure duration: 48 - 72 hoursNUMBER OF REPLICATIONS: 3 DETERMINATION OF CYTOTOXICITY- Method: other: decrease in the number of revertants and reduced background lawn
Evaluation criteria:
Generally, the experiment was considered valid if the following criteria were met:- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.- The sterility controls revealed no indication of bacterial contamination.- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.- Fresh bacterial culture containing approximately 109 cells per mL were used.The test substance was considered positive in this assay if the following criteria were met:- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.A test substance was generally considered non-mutagenic in this test if:- The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
TEST-SPECIFIC CONFOUNDING FACTORSNone.COMPARISON WITH HISTORICAL CONTROL DATA: In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.ADDITIONAL INFORMATION ON CYTOTOXICITY: No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate and in the preincubation test up to the highest required concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Standard plate test

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E.coli
  TA1535 TA1537 TA98 TA100 WP2 uvr A
     
  Results with S9
Spontaneous Reversion 8.3 10.0 36.7 49.3 61.0

Positive control

365.7 198.0 2178.7 2171.0 266.7
33 6.0 11.0 37.0 72.3 80.3
100 7.7 7.7 36.0 52.3 69.7
333 12.0 10.7 49.0 58.0 67.0
1000 9.7 10.0 41.0 61.0 64.7
2500 11.7 8.3 33.3 58.3 75.0
5000 11.0 14.0 25.0 57.7 80.3
           
  Results without S9

Spontaneous Reversion

8.3 6.7 25.0 40.0 63.0

Positive control

6320.7 2391.3 325.0 5669.7 893.3
33 9.7 5.0 23.7 35.0 60.3
100 9.7 7.3 25.3 37.7 70.3
333 7.0 7.7 28.0 42.7 58.3
1000 9.0 6.3 18.7 41.3 64.0
2500 8.7 5.0 26.3 33.3 62.0
5000 14.3 7.7 29.0 42.0 68.7

Table 2 Preincubation test

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E.coli
  TA1535 TA1537 TA98 TA100 WP2 uvr A
     
  Results with S9
Spontaneous Reversion 13.3 9.3 34.7 71.3 80.3

Positive control

240.3 90.3 645.0 2056.3 326.7
33 13.0 9.7 36.3 48.7 71.3
100 11.7 7.7 34.3 60.0 68.3
333 12.0 8.7 37.0 63.0 69.7
1000 11.0 10.0 30.7 61.3 68.3
2500 11.0 9.3 36.0 62.7 67.7
5000 10.3 8.7 38.3 54.7 67.0
           
  Results without S9
Spontaneous Reversion 10.3 8.3 23.0 44.7 60.0

Positive control

2329.0 1014.0 393.3 2797.3 368.7
33 9.3 8.0 25.7 44.3 68.3
100 8.7 7.3 25.3 44.3 66.7
333 9.3 6.7 25.7 49.0 57.7
1000 9.7 8.0 23.7 46.7 64.3
2500 8.3 8.0 26.3 44.3 57.0
5000 8.7 6.3 23.3 37.7 55.7

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negative