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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
Study period:
2010-02-01 to 2010-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
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): Alkenes, C11-12, hydroformylation products, distn. residues
- Substance type: pure active substance
- Physical state: turbid colourless liquid
- Storage condition of test material: in the dark at ambient room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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: 17 / 50 / 167 / 500 / 1667 / 5000 µg/plate
Main tests: 17 / 50 / 167 / 500 / 1667 / 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Initial solubility tests showed that acetone was a suitable solvent. Examination of the test item showed it to be a turbid liquid and it was normal for the suspended solid to separate from the liquid fraction at room temperature (confiremd by the Sponsor). The test item was thoroughly stirred before samples were taken for testing to ensure homogeneity. When the acetone was added, a clear, homogenous solution was obtained (both the solid and liquid components were fully in solution)
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-nitro-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 (direct plate method) in the first test; pre-incubation in the second (repeat) test


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


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 and for microcolony growth (condition of backgroun lawn was assessed as in the toxicity test).
- 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:
cytotoxicity
Remarks:
slight toxicity was observed at the highest concentration of 5000 µg/plate
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:
cytotoxicity
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: no data
- Precipitation: Precipitation at 1667 and 5000 µg/plate in both absence and presence of S9 mix.
- Other confounding effects: nothing mentioned


RANGE-FINDING/SCREENING STUDIES: Slight toxicity was observed as a thinning of the background lawn of microcolonies at the highest concentration in both the absence and 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 (Ames et al, 1975; Gatehouse et al, 1994). 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
 Acetone  77  -  97  -
17  86  1.1  87  0.9
 50  81  1.1  68  0.7
 167  76  1.0  60  0.6
 500  65  0.8  97  1.0
 1667  76 P  1.0  83 P  0.9
 5000  48 P ST  0.6  57 P ST  0.6

P = Precipitate / ST= Slightly Thin Lawn

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
Acetone 14.0 ± 5.3  -  18.7 ± 4.0  -  10.0 ± 2.6  -  10.0 ± 2.6  -  40.0 ± 11.1  -  45.0 ± 6.6  -
 17  13.0 ± 3.0  0.9  10.7 ± 1.2  0.6  5.7 ± 2.9  0.7  15.0 ± 5.0  1.5  32.0 ± 19.1  0.8  31.7 ± 3.2  0.7
 50  12.7 ± 2.5  0.9  14.3 ± 3.1  0.8  7.0 ± 3.0  0.7  16.3 ± 3.5  1.6  23.3 ± 4.7  0.7  38.3 ± 5.5  0.9
 167  15.3 ± 6.7  1.1  12.3 ± 4.6  0.7  11.0 ± 7.0  1.1  12.7 ± 1.5  1.3  31.3 ± 2.1  0.8  36.7 ± 12.9  0.8
 500  7.0 ± 3.5  0.5  15.0 ± 0.0  0.8  8.3 ± 6.0  0.8  10.3 ± 2.9  1.0  36.3 ± 18.6  0.9  33.0 ± 8.2  0.7
 1667  14.0 ± 4.4 P  1.0  18.0 ± 1.0 P  1.0  8.3 ± 2.1 P ST  0.8  6.7 ± 2.5 P  0.7  19.7 ± 3.2 P  0.5  33.3 ± 5.5 P  0.7
 5000  13.0 ± 4.0 P ST  0.9  16.0 ± 8.2 P  0.9  10.3 ± 2.3 P TL  1.0  7.3 ± 6.7 P ST  0.9   20.0 ± 5.3 P ST  1.0  33.7 ± 4.2 P ST  0.7
 Positive Control  470.0 ± 31.0  33.6  592.0 ± 42.5  31.7  5809.3 ± 170.5  581  361.7 ± 16.8  36.2  607.0 ± 21.0 15.2   822.7 ± 58.2  18.3

P = Precipitate / ST= Slightly Thin Lawn / TL = Thin Lawn

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
Acetone  91.7 ± 11.4  -  101.3 ± 6.4  -  8.7 ± 1.5  -  13.0 ± 2.6  -
 17  92.3 ± 7.4  1.0  88.0 ± 15.0  0.9  6.0 ± 2.6  0.7  10.7 ± 2.1  0.8
 50  89.7 ± 8.5  1.0  98.3 ± 22.5  1.0  7.7 ± 1.2  0.9  9.3 ± 3.1  0.7
 167  88.3 ± 5.0  1.0  79.7 ± 2.1  0.8 7.0 ± 0.0  0.8  8.0 ± 1.7  0.6
 500  93.7 ± 8.1  1.0  88.3 ± 4.0  0.9  5.7 ± 3.5  0.7  7.3 ± 1.5  0.6
 1667  80.0 ± 14.4 P  0.9  86.3 ± 7.4 P  0.9  7.0 ± 3.0 P  0.8  8.7 ± 3.5 P  0.7
 5000  72.0 ± 3.6 P ST  0.8  75.3 ± 3.1 P ST  0.7  6.7 ± 1.2 P ST  0.8  6.7 ± 3.1 P  0.5
 Positive control  1059.0 ± 60.6  11.6 1130.0 ± 46.7  11.2  130.3 ± 24.8  15.0  469.3 ± 14.3  36.1

P = Precipitate / ST = Slightly Thin Lawn

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
Acetone   12.7 ± 3.5  -  17.3 ± 4.5  -  9.0 ± 2.6  -  19.0 ± 2.6  -  27.0 ± 4.6  -  35.0 ± 4.4  -
 17  14.7 ± 8.5  1.2  18.3 ± 7.6  1.1  12.7 ± 2.1  1.4  20.7 ± 4.6  1.1  22.7 ± 8.5  0.8  37.7 ± 7.2  1.1
 50  10.0 ± 1.0  0.8  12.7 ± 3.8  0.7  11.3 ± 2.1  1.3  20.0 ± 4.0  1.1  23.0 ± 5.0  0.9  39.7 ± 6.0  1.1
 167  14.3 ± 5.1  1.1  14.3 ± 5.9  0.8  14.7 ± 4.6  1.6  16.0 ± 2.6  0.8  20.7 ± 1.2  0.8  33.0 ± 2.0  0.9
 500  15.3 ± 4.9  1.2  18.0 ± 2.0  1.0  11.3 ± 2.1  1.3  12.7 ± 3.1  0.7  27.3 ± 9.0  1.0  31.7 ± 4.9  0.9
 1667  13.7 ± 2.1P ST  1.1  12.0 ± 5.6 P  0.7  8.7 ± 4.0 P TL  1.0  4.3 ± 2.1 P ST  0.2  19.7 ± 3.2 P TL  0.7  27.3 ± 4.0 P  0.8
 5000  10.7 ± 4.0 P TL  0.8  13.3 ± 3.2 P ST  0.8  5.3 ± 0.6 P TL  0.6  9.0 ± 2.6 P TL  0.5  24.5 P TL  0.9  32.3 ± 9.8 P ST  0.9
 Positive Control  525.0 ± 20.0  41.4  317.7 ± 13.7  18.3 4462.7 ± 423.7  496  179.0 ± 29.5  9.4  377.3 ± 28.1  14.0  177.7 ± 15.3  5.1

P = Precipitate / ST = Slightly Thin Lawn / TL = Thin Lawn

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
 Acetone  100.7 ± 10.5  -  95.7 ± 20.6  -  11.0 ± 1.7 - 9.7 ± 5.5  -
 17  78.7 ± 16.2  0.8  78.7 ± 5.8  0.8  8.3 ± 6.1  0.8  7.3 ± 2.5  0.8
 50  69.7 ± 4.7  0.7  94.3 ± 11.8  1.0  9.0 ± 3.6  0.8  6.3 ± 4.0  0.7
 167  74.0 ± 19.1  0.7  100.3 ± 7.6  1.0  12.7 ± 3.5  1.2  3.3 ± 1.5  0.3
 500  72.7 ± 5.0  0.7  88.0 ± 7.8  0.9  5.7 ± 0.6  0.5  5.7 ± 1.5  0.6
 1667  73.7 ± 6.1 P VT  0.7  85.3 ± 8.1 P  0.9  2.7 ± 1.2 P TL  0.2  5.3 ± 1.5 P  0.6
 5000  67.0 ± 7.0 P TL  0.7  88.3 ± 4.5 P ST  0.9  2.0 P VT  0.2  8.0 ± 2.6 P  0.8
 Positive control  1112.0 ± 33.5  11.0  570.7 ± 61.4  6.0  186.0 ± 36.5  16.9  242.3 ± 6.4  25.1

P = Precipitate / ST = Slightly Thin Lawn / TL = Thin Lawn / VT = Very Thin Lawn

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.
In the direct plate incorporation test, toxicity was encountered in all stains at the highest concentration or 2 highest concentrations in the absence of S9 mix and in 3 of 5 strains at the highest concentration in the presence of S9 mix. In the pre-incubation test, toxicity was encountered at the 2 highest concentrations in all strains in the absence of S9 mix, and in 4 of the 5 strains at the highest concentration or 2 highest concentrations in the presence of S9 mix. The test item precipitated at concentration s 1667 and 5000 µg/plate in all tests. In addition, the test substance precipitated at 1667 and 5000 µg/plate.
Executive summary:

Alkenes, C11 -12 hydroformylation products, 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 dissolved and diluted in Acetone. 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 17 to 5000 µg/plate in both assays. The highest concentration was the predetermined maximum, as recommended by relevant guidelines, but was in addition both toxic to the bacteria and above the limit of solubility of the tes item in the test system.

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.

In the direct plate incorporation test, toxicity was encountered in all stains at the highest concentration or 2 highest concentrations in the absence of S9 mix and in 3 of 5 strains at the highest concentration in the presence of S9 mix. In the pre-incubation test, toxicity was encountered at the 2 highest concentrations in all strains in the absence of S9 mix, and in 4 of the 5 strains at the highest concentration or 2 highest concentrations in the presence of S9 mix. The test item precipitated at concentration s 1667 and 5000 µg/plate in all tests.

It was concluded that Alkenes, C11 -12, hydroformylation products, distn. residues was not mutagenic in strains of Salmonella typhimurium and Escherichia coli when tested in acetone in the absence and presence of metabolic activation. The test item was tested to the predetermined maximum of 5000 µg per plate, at which concentration toxicity was encountered. In addition, the test item was tested up to and beyond its limits of solubility in the test system.

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