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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Alcohols, C18-22, distn. residues
Cas Number:
1160164-88-4
IUPAC Name:
Alcohols, C18-22, distn. residues
Details on test material:
- Name of test material (as cited in study report): Alcohols, C18-22 Distn. Residues
- Substance type: pure active substance
- Physical state: solid
- Storage condition of test material: in the dark at ambient room temperature

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 enzymes from the livers of Aroclor 1254-treated adult, male Fisher rats
Test concentrations with justification for top dose:
Toxicity test: 6 / 20 / 60 / 200 / 600 / 2000 µg/plate
Main tests: 6 / 20 / 60 / 200 / 600 / 2000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In preliminary tests the test item was found to have limited solubility in all solvents compatibel with the test system. DMSO was chosen as the solvent giving the best solubility/dispersal characteristics.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 1 µg/plate with S. typhimurium TA 1535 and TA100; 9-Aminoacridine, 80 µg/plate with S. typhimurium TA 1537; 2-Nitrofluorene, 1 µg/plate with S. typhimurium TA 98; N-Ethyl-N-nitrosoguanidine, 2 µg/plate with E. coli WP2uvrA
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, 2 µg/plate with S. typhimurium TA 1535 and TA 1537, 0.5 µg/plate with S. typhimurium TA 98 and TA 100, 20 µg/plate with E. coli WP2uvrA
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) in the first test; preincubation in the second (repeat) test


DURATION
- Preincubation period: 20 min (only in the repeat test)
- Exposure duration: 2 - 3 days


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY:
- Method: Plates were microscopically examined for thinning of background lawn, condition of background lawn was assessed as normal, slightly thin lawn, thin lawn, very thin lawn or lawn absent.


OTHER EXAMINATIONS:
- The numbers of mutant colonies on each plate were determined using a Sorcerer Colony Counter (Perceptive Instruments) and captured electronically in a validated software system (Ames Study Manager, Perceptive Instruments), the plates were also examined microscopically for precipitates.
- Quality control of bacterial strains: All bacterial strains were tested for ampicillin resistence, crystal violet and ultraviolett radiation sensitivity and for essential animo acid requirement.

OTHER:
To establish suitable exposure levels for the first mutation test an initial dose-finding test in the presence and absence of S9 mix with a single strain of bacteria, S. typhimurium TA 100 and one plate per exposure level was conducted (Toxicity test).
Evaluation criteria:
Interpretation of mutagenicity:
- doubling of the mean concurrent vehicle control value for S. typhimurium strains TA 1535, TA 1537 and TA 98 and for E. coli WP2uvrA, resp. 1.5-fold increase over the control value for S. typhimurium strain TA 100
- if the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes (in such cases a minimum count of 20, representing a 2-fold increase over 10, was required before a response was registered)
- concentration-related response, at high concentration, this relationship may be reversed
- a response should be reproducible in the independent test
Acceptance criteria:
- each bacterial strain demonstrated typical responses to crystal violet, ampicillin and ultralviolet radiation
- at least 2 of the 3 vehicle control plates were within the historical vehicle control data
- at least 2-fold increases over the mean vehicle control values in at least 2 of the 3 positive control plates for each strain and activation state were obtained (in the case of TA 100, at least 1.5-fold was obtained)
- no toxicity or contamination was observed in at least 4 concentration levels
- in cases where a mutagenic response was observed, no more than one exposure level was discarded below the concentration that gave the highest mean colony number
Statistics:
not performed

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
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
other: not tested
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
other: not tested
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: poorly soluble in water
- Precipitation: Precipitated/undissolved test item was observed at the highest test concentration (2000 µg/plate) in both absence and presence of S9 mix.
- Other confounding effects: nothing mentioned


RANGE-FINDING/SCREENING STUDIES: No toxicity to the bacteria was observed at the highest concentration of 2000 µg/plate in either the absence or the presence of S9 mix.


COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control values were within the normal/historical ranges recorded in the testing laboratory and reported in the literature with these strains of S. typhimurium and E. coli. The positive control values were also within the normal/historical ranges for each bacterial strain and activation condition.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table #1: Toxicity test with S. typhimurium strain TA 100

      without metabolic activation with metabolic activation    
Dose level [µg/plate]   Revertant colony count  Ratio treated/solvent  Revertant colony count  Ration treated/solvent
 DMSO  84  -  110  -
 65  0.8  92  0.8
 20  95  1.1  99  0.9
 60  116  1.4  103  0.9
 200  88  1.0  87  0.8
 600  95  1.1  84  0.8
 2000  79 P  0.9  104 P  0.9

P = Precipitate

Table #2: First Mutation Assay (Direct Plate Incorporation Method)

  TA 1535           TA 1537               TA 98      
      - S9 mix + S9 mix     - S9 mix   + S9 mix     - S9 mix     + S9 mix    
Dose level[µg/plate]   Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertantsper plate± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio
DMSO  18.3 ± 8.1   -  16.3 ± 0.6  -  15.3 ± 8.6  -  16.7 ± 1.5  -  28.3 ± 11.9  -  32.0 ± 6.9  -
 6  15.0 ± 3.5  0.8  14.0 ± 2.6  0.9  10.3 ± 5.1  0.7  8.3 ± 1.2  0.5  27.0 ± 5.6  1.0  37.3 ± 11.5  1.2
 20  12.3 ± 3.1  0.7  16.3 ± 6.0  1.0  11.0 ± 1.0  0.7  17.0 ± 10.0  1.0  22.7 ± 1.5  0.8  37.3 ± 7.8  1.2
 60  17.7 ± 6.7  1.0  15.7 ± 4.0  1.0  9.0 ± 2.6  0.6  16.0 ± 5.0  1.0  25.3 ± 5.1  0.9  42.3 ± 10.4  1.3
 200  15.3 ± 2.5  0.8  11.7 ± 6.5  0.7  13.3 ± 7.5  0.9  15.0 ± 2.0  0.9  27.7 ± 2.9  1.0  33.3 ± 9.5  1.0
 600  23.3 ± 4.0  1.3  18.7 ± 8.1  1.1  10.0 ± 3.0  0.7  15.7 ± 2.3  0.9  26.0 ± 4.4  0.9  44.7 ± 1.5  1.4
 2000  15.7 ± 2.3 P  0.9  17.0 ± 2.6 P  1.0  12.3 ± 2.3 P  0.8  13.0 ± 5.2 P  0.8  23.5 ± 10.6 P  0.8  41.7 ± 2.1 P  1.3
 Positive Control  554.3 ± 35.0  30.2  629.0 ± 28.2  38.5  4833.7 ± 346.1  315  434.7 ± 45.2  26.1  880.0 ± 19.7  31.1  509.3 ± 66.5  15.9

Table #2 (continued): First Mutation Assay (Direct Plate Incorporation Method)

  TA 100           WP2uvrA          
  - S9 mix     + S9 mix     - S9 mix      + S9 mix   
Dose level [µg/plate]   Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio
 DMSO  88.0 ± 11.1  -  96.7 ± 8.1  -  7.0 ± 3.0  -  13.3 ± 1.5  -
 6  95.3 ± 19.6  1.1  95.0 ± 9.8  1.0  10.0 ± 3.0  1.4  13.7 ± 2.9  1.0
 20  102.0 ± 3.5  1.2  102.3 ± 12.5  1.1  10.3 ± 3.1  1.5  5.0 ± 0.0  0.4
 60  103.0 ± 2.0  1.2  106.3 ± 12.9  1.1  6.3 ± 2.5  0.9  10.7 ± 2.1  0.8
 200  100.7 ± 3.5  1.1  103.0 ± 5.3  1.1  5.3 ± 1.5  0.8  7.0 ± 2.0  0.5
 600  93.3 ± 7.0  1.1  114.3 ± 10.3  1.2  10.7 ± 4.7  1.5  10.0 ± 1.0  0.8
 2000  104.0 ± 19.7 P  1.2  104.7 ± 6.4 P  1.1  3.3 ± 2.3 P  0.5  6.3 ± 2.3 P  0.5
 Positivecontrol  989.7 ± 45.1  11.2  1093.3 ± 72.2  11.3  90.3 ± 17.9  12.9  519.7 ± 13.6  39.0

Table #3: Second Mutation Assay (Pre-incubation Method)

  TA 1535           TA 1537               TA 98      
      - S9 mix + S9 mix     - S9 mix   + S9 mix     - S9 mix     + S9 mix    
Dose level [µg/plate]   Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertantsper plate± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio
DMSO   10.3 ± 2.9  -  11.7 ± 1.5  -  12.7 ± 2.5  -  10.3 ± 2.3  -  25.7 ± 1.5  -  30.0 ± 3.6  -
 6  12.0 ± 4.6  1.2  10.3 ± 1.5  0.9  10.3 ± 1.2  0.8  10.0 ± 5.2  1.0  25.3 ± 3.8  1.0  23.7 ± 3.1  0.8
 20  10.3 ± 0.6  1.0  11.7 ± 0.6  1.0  10.7 ± 4.0  0.8  10.3 ± 4.5  1.0  18.7 ± 4.7  0.7  24.0 ± 2.0  0.8
 60  13.3 ± 3.2  1.3  18.0 ± 1.7  1.5  10.3 ± 1.2  0.8  18.0 ± 2.6  1.7  26.7 ± 2.5  1.0  37.7 ± 4.5  1.3
 200  15.7 ± 2.5  1.5  15.7 ± 4.5  1.3  11.0 ± 2.0  0.9  12.0 ± 5.6  1.2  28.3 ± 8.5  1.1  23.7 ± 2.1  0.8
 600  13.7 ± 2.9  1.3  15.7 ± 6.0  1.3  12.0 ± 6.6  0.9  10.3 ± 1.5  1.0  22.7 ± 8.3  0.9  32.3 ± 4.6  1.1
 2000  14.0 ± 6.1 P  1.4  18.0 ± 2.0 P  1.5  15.0 ± 3.0 P  1.2  11.7 ± 5.7 P  1.1  21.0 ± 2.6 P  0.8  31.3 ± 2.9 P  1.0
 Positive Control  513.7 ± 37.8  49.7  368.7 ± 25.3  31.6  4223.3 ± 423.4  333  224.3 ± 40.7  21.7  608.0 ± 90.8  23.7  697.7 ±33.3  23.3

Table #3 (continued): Second Mutation Assay (Pre-incubation Method)

  TA 100           WP2uvrA          
  - S9 mix     + S9 mix     - S9 mix      + S9 mix   
Dose level [µg/plate]   Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio
 DMSO  88.3 ± 2.1  -  109.3 ± 12.0  -  4.7 ± 4.0 - 9.3 ± 3.1  -
 6  76.3 ± 11.9  0.9  96.0 ± 15.6  0.9  8.3 ± 1.2  1.8  13.7 ± 4.2  1.5
 20  89.0 ± 19.3  1.0  96.7 ± 8.6  0.9  8.7 ± 3.2  1.9  8.0 ± 6.1  0.9
 60  97.3 ± 25.8  1.1  104.0 ± 6.0  1.0  13.3 ± 3.8  2.9  10.3 ± 1.5  1.1
 200  91.7 ± 5.5  1.0  103.0 ± 6.1  0.9  6.0 ± 2.6  1.3  13.0 ± 2.6  1.4
 600  101.3 ± 14.6  1.1  100.0 ± 5.6  0.9  6.0 ± 1.0  1.3  15.7 ± 6.0  1.7
 2000  90.0 ± 9.8 P  1.0  96.0 ± 3.5 P  0.9  9.7 ± 0.6 P  2.1  9.7 ± 1.5 P  1.0
 Positive control  1082.0 ± 55.1  12.2  843.7 ± 67.5  7.7  153.3 ± 23.0  32.9  469.3 ± 26.3  50.3

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

No evidence of mutagenic activity was obtained with any strain in either test.
There was no toxicity to any of the strains of bacteria in either test.
Precipitated/undissolved test item was observed on the plates at 2000 µg/plate in both the absence and the presence of S9 mix in both tests.
Executive summary:

Alcohols, C18 -22 Distn. Residues was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA100 and in Escherichia coli WP2uvrA according to OECD guideline 471and the European Commision Annex V Test Method B13 and B14.

The test item was formulated in Dimethylsulfoxide, in which it gave a part solution/part suspension. Two independent tests were conducted on agar plate in triplicate in the absence and presence of an Aroclor 1254 -induced rat liver S9 preparation and co-factors required for mixed function oxidase activity (S9 mix). The first test was conducted by the direct plate incorporation method, while the second test was conducted by the pre-incubation method. The test item was dosed at concentrations ranging from 6 to 2000 µg/plate in both assay (2000 µg/plate was the highest practical concentration, as limited by the solubility of the test item).

Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix.

No evidence of mutagenic activity was obtained with any strain in either test.

No toxicity of the bacteria was observed. The test item precipitated at the highest concentration of 2000 µg/plate.

It was concluded that Alcohols, C18 -22 Distn. Residues was not mutagenic in strains of Salmonella typhimurium and Escherichia coli, when tested in the absence and presence of metabolic activation up to and beyond its limit of solubility in the test system.

The study was performed in accordance with the principles of Good Laboratory Practice.