Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
A bacterial reverse mutation assay (Ames test) was conducted in different bacterial strains. The test substance was found to be non mutagenic with and without metabolic activation but cytotoxic to the bacteria in high concentrations.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-12-19 to 2001-01-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium strains had mutations in the histidine locus (histidine deficiency), rfa-minus (deficient lipopolysaccharide envelope), uvrB-minus (deficient excision repair) and TA 98, TA 100 and TA 102 carried R-factor plasmid pKM 101 (ampicillin resistance marker). The histidine mutation of TA 102 was unrevertable but a revertable histidine mutation in the pAQ1 plasmid. This allowed the detection of A-T base mutations in contrast to G-C base mutations detected by TA 1535 and TA 100. TA 1537 and TA 98 detected frameshift mutations.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
1.5, 5, 15, 50, 150 and 500 µg/plate
Vehicle / solvent:
- Vehicle: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for TA98 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA1537 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA100 and TA1535 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
for TA102 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
for TA98, TA100, TA102, TA1535 und TA1537 with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertant colonies

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

COMPARISON WITH HISTORICAL CONTROL DATA: data was stated but no direct comparison was made

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without metabolic activation cytotoxicity was observed for all test strains at a concentration of 150µg/plate. With metabolic activation cytotoxicity was detected for 150µg/plate only in TA 102. The other test strains showed cytotoxicity with metabolic activation at 500 µg/plate.

Experiment 1 (plate incorporation)

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
TA98 TA100 TA102 TA1535 TA1537
Results without S9
Untreated 32± 3 97 ± 4 285 ± 14 23 ± 6 10 ± 4
DMSO 35 ± 3 95 ± 6 247 ± 13 19 ± 4 12 ± 2
5 34 ± 4 103 ± 10 254 ± 22 32 ± 10 11 ± 2
15 40 ± 3 92 ± 13 248 ± 18 21 ± 9 14 ± 3
50 35 ± 5 86 ± 5 249 ± 10 14 ± 6 10 ± 3
150 7 ± 2 68 ± 4 0 ± 0 13 ± 4 6 ± 3
500 0 ± 1 14 ± 16 0 ± 0 0 ± 0 1 ± 2
NaN3 (0.7) 409 ± 17 804 ± 17
2-NF (2.5) 402 ± 35
9-AA (50) 300 ± 17
Mitomycin C (0.15) 604 ± 50
Results with S9
Untreated 22 ± 5 124 ± 14 340 ± 25 11 ± 4 14 ± 4
DMSO 20 ± 2 98 ± 5 320 ± 22 8 ± 1 15 ± 3
5 22 ± 3 105 ± 2 264 ± 25 7 ± 3 13 ± 4
15 20 ± 3 103 ± 6 297 ± 18 7 ± 2 16 ± 3
50 17 ± 3 112 ± 4 267 ± 13 6 ± 2 14 ± 3
150 16 ± 4 104 ± 17 172 ± 19 6 ± 3 12 ± 3
500 5 ± 4 69 ± 9 30 ± 26 1 ± 1 9 ± 3
2-AA (0.8) 294 ± 7 429 ± 27 431 ± 21 103 ± 14
2-AA (1.7) 224 ± 22

Experiment 2 (plate incorporation)

Dose (µg/plate) Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
TA98 TA100 TA102 TA1535 TA1537
Results without S9
Untreated 41 ± 1 92 ± 5 275 ± 18 18 ± 3 13 ± 5
DMSO 36 ± 2 85 ± 8 257 ± 18 23 ± 4 16 ± 0
1.5 33 ± 4 82 ± 4 268 ± 10 18 ± 3 12 ± 2
5 33 ± 11 85 ± 6 277 ± 23 19 ± 4 18 ± 3
15 35 ± 2 71 ± 12 235 ± 30 17 ± 5 15 ± 5
50 28 ± 7 70 ± 10 216 ± 22 21 ± 4 13 ± 3
150 8 ± 2 5 ± 5 0 ± 0 0 ± 0 1 ± 2
NaN3 (0.7) 296 ± 20 798 ± 168
2-NF (2.5) 581 ± 25
9-AA (50) 305 ± 24
Mitomycin C (0.15) 685 ± 46
Results with S9
Untreated 17 ± 4 91 ± 8 295 ± 32 14 ± 4 18 ± 4
DMSO 14 ± 2 85 ± 2 271 ± 11 10 ± 1 15 ± 2
1.5 10 ± 2 303 ± 23 8 ± 3 13 ± 7
5 13 ± 4 90 ± 7 278 ± 13 8 ± 3 12 ± 2
15 13 ± 5 91 ± 4 293 ± 23 7 ± 2 14 ± 4
50 13 ± 5 94 ± 5 274 ± 12 8 ± 3 12 ± 5
150 9 ± 3 72 ± 11 234 ± 30 7 ± 2 13 ± 3
500 0 ± 0
2-AA (0.8) 211 ± 16 352 ± 71 434 ± 69 130 ± 7
2-AA (1.7) 143 ± 44
Conclusions:
A bacterial reverse mutation assay (Ames test) was conducted in different bacterial strains. The test substance was found to be non mutagenic with and without metabolic activation but cytotoxic to the bacteria in high concentrations.
Executive summary:

The mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102) according to OECD 471. The investigations were carried out using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations of 1.5 to 500 µg per plate in the presence and absence of S9. In the absence of S9-mix the test substance was bacteriotoxic towards all strains at 150 µg/plate. In the presence of S9-mix the test substance was bacteriotoxic towards the strains TA102 at 150 µg /plate and towards the strains TA1535, TA1537, TA98, and TA100 at 500 μg/plate. Precipitation of the test compound on the plates was not observed.

Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.

In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A bacterial reverse mutation assay (Ames test) was conducted to study the mutagenicity of the test substance with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102) according to OECD 471. The investigations were carried out using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test substance was dissolved in DMSO and tested in concentrations of 1.5 to 500 µg per plate in the presence and absence of S9. In the absence of S9-mix the test substance was bacteriotoxic towards all strains at 150 µg/plate. In the presence of S9-mix the test substance was bacteriotoxic towards the strains TA102 at 150 µg /plate and towards the strains TA1535, TA1537, TA98, and TA100 at 500 μg/plate. Precipitation of the test compound on the plates was not observed. Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.