Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2013-12-13 to 2014-06-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: the study was conducted under GLP and according to OECD 471 (1997), EPA OPPTS 870.5100 (1998) and EC 440/2008 B.13/14 (2008) without deviations.

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4,6-trichloroaniline
EC Number:
211-219-8
EC Name:
2,4,6-trichloroaniline
Cas Number:
634-93-5
Molecular formula:
C6H4Cl3N
IUPAC Name:
2,4,6-trichloroaniline
Test material form:
not specified
Details on test material:
- Storage condition of test material: Stored at room temperature

Method

Target gene:
The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his-/his+ and trp-/trp+ reversions, respectively. The S. typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA1535, TA100, WP2 uvrA pKM101, and WP2 pKM101) and frameshift (TA1537, TA98) mutations.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: tryptophan biosynthesis, DNA repair process
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 – 12 w old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands).
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test substance was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I as at least five concentrations were analysable. Since toxic effects were solely observed at high concentrations, 5000 µg/plate was also chosen as maximal concentration of the second experiment.
The concentration range included two logarithmic decades. The following concentrations were tested in experiment II:
3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (purity 99 %)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: direct plate incorporation and pre-incubation method

DURATION
- Preincubation period: 60 minutes
For the pre-incubation method 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer (7 parts of the 100 mM sodium-ortho-phosphate-buffer pH 7.4 with 3 parts of KCl solution 0.15 M) and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for 72 hours at 37°C in the dark, plates were then stored at 4°C until counted.
- Exposure duration: 60 minutes (pre-exposure) + 72 hours (exposure)
- Expression time (cells in growth medium): in the pre-culture 10^8 to 10^9 cells/mL
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: number of revertant colonies

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
Evaluation criteria:
Acceptability of the assay
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below an induction factor of 0.5.

Evaluation of results
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: DMSO was used as solvent.
- Precipitation: The test substance precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with and without S9 mix in both experiments. Precipitation of the test substance in the overlay agar was also observed on the incubated agar plates from 1000 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II. The undissolved particles had no influence on the data recording.
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
To evaluate the cytotoxicity of the test substance a pre-experiment was performed with all strains. Eight concentrations were tested for cytotoxicity and mutation induction each with three replicate plates. The experimental conditions in this pre-experiment were the same as described below for experiment I (plate incorporation test).
Cytotoxicity of the test substance results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as the main experiment I since the criteria mentioned under Acceptability of the assay were met.

COMPARISON WITH HISTORICAL CONTROL DATA:
The laboratory historical control range was not quite reached in the untreated control of strain WP2 uvrA pKM101 with S9 mix in experiment I. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Reduced background growth was observed at the following concentrations (µg/plate):
TA 1535: Experiment I (without S9 mix): 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
TA 1537: Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 1000 - 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
TA 98: Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
TA 100: : Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 1000 - 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
WP2 pKM101: : Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
WP2 uvrA pKM101: : Experiment I (without S9 mix): 2500, 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000

Toxic effects, evident as a reduction in the number of revertants (below an induction factor of 0.5), were observed at the following concentrations (µg/plate):
TA 1535: Experiment I (without S9 mix): / --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 5000 --- Experiment I (with S9 mix): 2500, 5000
TA 1537: Experiment I (without S9 mix): 5000 --- Experiment I (with S9 mix): 1000 - 5000 --- Experiment II (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 333 - 5000
TA 98: Experiment I (without S9 mix): 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 333 - 5000
TA 100: Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 1000 - 5000 --- Experiment II (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 333 - 5000
WP2 pKM101: Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment I (with S9 mix): 333 - 5000
WP2 uvrA pKM101: Experiment I (without S9 mix): 2500, 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 333 - 5000
/ = no toxic effects observed


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Summary of Results Pre-Experiment/Experiment I

Metabolic Activation Test group Concentration (per plate) Revertant Colony Counts (Mean ±SD)
TA 1535 TA 1537 TA 98 TA 100 WP2 pKM101 WP2 uvrA pKM101
Without Activation DMSO --- 16 ± 1 14 ± 6 22 ± 4 91 ± 3 228 ± 24 327 ± 30
Untreated --- 11 ± 2 13 ± 2 26 ± 6 100 ± 15 249 ± 12 373 ± 1
test item 3 µg 13 ± 4 9 ± 1 20 ± 3 91 ± 9 231 ± 23 331 ± 29
10 µg 16 ± 3 12 ± 2 23 ± 7 85 ± 10 209 ± 16 334 ± 46
33 µg 18 ± 1 10 ± 2 19 ± 7 91 ± 5 190 ± 18 294 ± 37
100 µg 19 ± 5 14 ± 1 25 ± 1 72 ± 5 224 ± 24 312 ± 42
333 µg 14 ± 3 12 ± 6 23 ± 3 54 ± 17 174 ± 26 311 ± 19
1000 µg 20 ± 3 12 ± 4P R  16 ± 1P R  31 ± 6P M R  61 ± 4P R  205 ± 41
2500 µg 14 ± 3P M  6 ± 3P R M  11 ± 3P M R  24 ± 1P M R  33 ± 4P M R  93 ± 9P M R 
5000 µg 9 ± 2P M R  5 ± 2P R M  4 ± 1P M R  16 ± 4P M R  26 ± 3P M R  58 ± 12P M R 
NaN3 10 µg 2919 ± 285 --- --- 2303 ± 99 --- ---
4-NOPD 10 µg --- --- 388 ± 12 --- --- ---
4-NOPD 50 µg --- 58 ± 5 --- --- --- ---
MMS 2.0 µL --- --- --- --- 3663 ± 93 4303 ± 35
With Activation DMSO --- 15 ± 3 22 ± 6 32 ± 3 100 ± 8 217 ± 2 366 ± 5
Untreated --- 16 ± 2 20 ± 6 35 ± 2 116 ± 16 269 ± 35 373 ± 43
test item 3 µg 16 ± 4 25 ± 4 33 ± 2 101 ± 14 243 ± 19 349 ± 14
10 µg 13 ± 1 17 ± 2 34 ± 7 105 ± 6 192 ± 11 316 ± 28
33 µg 15 ± 6 17 ± 3 35 ± 8 114 ± 12 201 ± 15 295 ± 21
100 µg 17 ± 1 14 ± 7 34 ± 4 103 ± 8 203 ± 58 304 ± 8
333 µg 14 ± 1 20 ± 5 30 ± 7 83 ± 16 192 ± 19 310 ± 58
1000 µg 11 ± 1 9 ± 2P M R  23 ± 6 42 ± 6P M R  104 ± 17 212 ± 21
2500 µg 3 ± 1P M R  5 ± 2P M R  8 ± 2P M R  16 ± 3P M R  24 ± 3P M R  98 ± 16P M R 
5000 µg 2 ± 1P M R  1 ± 1P M R  1 ± 1P M R  0 ± 1P M R  4 ± 2P M R  12 ± 2P M R 
2-AA 2.5 µg 498 ± 40 349 ± 36 1852 ± 77 2408 ± 268 --- ---
2-AA 10.0 µg --- --- --- --- 3552 ± 129 1822 ± 155

NaN3. sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

P: Precipitate

M: Manual count

R: Reduced background growth

Table 2 Summary of Results Experiment II

Metabolic Activation Test group Concentration (per plate) Revertant Colony Counts (Mean ±SD)
TA 1535 TA 1537 TA 98 TA 100 WP2 pKM101 WP2 uvrA pKM101
Without Activation DMSO --- 16 ± 5 9 ± 2 27 ± 4 96 ± 17 243 ± 2 407 ± 11
Untreated --- 17 ± 4 10 ± 5 32 ± 1 105 ± 7 276 ± 27 419 ± 6
test item 3 µg 15 ± 1 9 ± 3 19 ± 5 93 ± 16 247 ± 12 412 ± 23
10 µg 13 ± 6 8 ± 1 17 ± 2 83 ± 11 232 ± 17 413 ± 10
33 µg 14 ± 2 11 ± 1 16 ± 3 91 ± 10 235 ± 11 460 ± 10
100 µg 19 ± 5 6 ± 2 21 ± 2 72 ± 14 112 ± 31 259 ± 15
333 µg 16 ± 3P R  6 ± 1P R  17 ± 4P R  59 ± 10P R  68 ± 7P R  207 ± 16P R 
1000 µg 14 ± 5P M R  3 ± 1P M R  9 ± 2P M R  13 ± 5P M R  32 ± 7P M R  112 ± 6P M R 
2500 µg 8 ± 3P M R  3 ± 2P M R  2 ± 1P M R  21 ± 1P M R  15 ± 4P M R  47 ± 5P M R 
5000 µg 3 ± 2P M R  0 ± 0P M R  0 ± 0P M R  0 ± 0P M R  8 ± 4P M R  17 ± 4P M R 
NaN3 10 µg 2706 ± 139 --- --- 2241 ± 83 --- ---
4-NOPD 10 µg --- --- 341 ± 25 --- --- ---
4-NOPD 50 µg --- 75 ± 16 --- --- --- ---
MMS 2.0 µL --- --- --- --- 3107 ± 175 2908 ± 14
With Activation DMSO --- 11 ± 4 15 ± 1 36 ± 2 104 ± 12 266 ± 23 453 ± 3
Untreated --- 10 ± 3 17 ± 4 31 ± 9 131 ± 5 314 ± 22 449 ± 1
test item 3 µg 7 ± 4 19 ± 7 33 ± 4 106 ± 13 262 ± 5 431 ± 49
10 µg 12 ± 2 11 ± 3 29 ± 5 115 ± 1 252 ± 5 444 ± 7
33 µg 14 ± 2 10 ± 4 26 ± 3 95 ± 18 267 ± 21 486 ± 6
100 µg 15 ± 6 13 ± 5 37 ± 8 105 ± 3 256 ± 19 424 ± 28
333 µg 8 ± 5P M R  3 ± 5P M R  16 ± 5P M R  23 ± 6P M R  32 ± 9P M R  78 ± 9P M R 
1000 µg 8 ± 4P M R  0 ± 1P M R  4 ± 1P M R  3 ± 2P M R  25 ± 6P M R  50 ± 11P M R 
2500 µg 2 ± 1P M R  4 ± 1P M R  0 ± 0P M R  0 ± 0P M R  2 ± 1P M R  21 ± 2P M R 
5000 µg 1 ± 1P M R  0 ± 0P M R  0 ± 0P M R  0 ± 0P M R  0 ± 1P M R  0 ± 0P M R 
2-AA 2.5 µg 408 ± 5 327 ± 12 2406 ± 113 2974 ± 707 --- ---
2-AA 10.0 µg --- --- --- --- 2072 ± 44 2205 ± 126

NaN3. sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

P: Precipitate

R: reduced background growth

M: Manual count

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test and the pre-incubation test using the Salmonella typhimurium strains and the Escherichia coli strains. The plates incubated with the test substance showed reduced background growth at higher concentrations with all strains used. Cytotoxic effects were observed in all strains at higher concentrations with the exception of strain TA 1535 in the first experiment without metabolic activation. No increase in revertant colony numbers of any of the six tester strains was observed. 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 of induced revertant colonies. The test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.