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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No evidence of mutagenic activity was obtained with any in vitro tests.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material: Alcohols, C2-33, manuf. of, by-products from overheads
- Substance type: Product, HF-1000
- Physical state: clear yellow liquid
- Odour: oily/solvent
- Purity test date: 2010/10/24
- Lot/batch No.: 68310
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. It was therefore used to formulate the test substance throughout the study.
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 minimum count of 20 is (representing a 2-fold increase over 10) is required before a response is 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
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
other: not tested
Positive controls validity:
valid
Key result
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 500, 1667 and 5000 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 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 (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'.

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  87  -  95  -
17  62  0.7  87  0.9
 50  81  0.9  112  1.2
 167  78  0.9  81  0.9
 500  79 P  0.9  79 P  0.8
 1667  75 P  0.9  112 P  1.2
 5000  75 P  0.9  66 P  0.7

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
Acetone 11.3 ± 2.1  -  13.7 ± 7.8  -  9.7 ± 3.1  -  7.3 ± 3.2  -  17.0 ± 5.6  -  33.0 ± 10.4  -
 17  7.7 ± 4.9  0.7  11.0 ± 3.5  0.8  10.7 ± 1.2  1.1  13.0 ± 3.6  1.8  18.3 ± 3.5  1.1  34.0 ± 7.0 1.0
 50  14.3 ± 2.9  1.3  8.7 ± 2.9  0.6  12.0 ± 1.0  1.2  8.0 ± 4.6  1.1  20.7 ± 5.5  1.2  31.3 ± 2.1  0.9
 167  8.7 ± 2.9  0.8  11.0 ± 3.5  0.8  7.7 ± 4.6  0.8  13.0 ± 1.7  1.8  22.7 ± 7.6  1.3  29.3 ± 4.2  0.9
 500  10.0 ± 6.6 P  0.9  11.7 ± 3.8 P  0.9  6.7 ± 2.5 P  0.7  12.7 ± 2.1 P  1.7  19.3 ± 6.4 P  1.1  26.7 ± 5.9 P  0.8
 1667  6.3 ± 5.1 P  0.6  11.0 ± 4.6 P  0.8  7.3 ± 2.3 P  0.8  14.0 ± 4.6 P  1.9  24.0 ± 8.5 P  1.4  27.3 ± 5.1 P  0.8
 5000  8.3 ± 2.9 P  0.7  14.0 ± 4.6 P  1.0  12.7 ± 3.1 P  1.3  13.3 ± 3.2 P  1.8  23.7 ± 8.3 P  1.4  30.3 ± 2.3 P  0.9
 Positive Control  444.0 ± 49.0  39.2  331.0 ± 26.5  24.2  3764.0 ± 423.2  389.4  272.0 ± 14.1  37.1  918.7 ± 184.7 54.0   365.0 ± 12.8  11.1

P = Precipitate

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 92.7 ± 5.5  -  93.0 ± 18.5  -  6.7 ± 4.5  -  8.0 ± 3.5  -
 17  91.3 ± 11.4  1.0  82.3 ± 13.6  0.9  7.7 ± 4.7  1.1  11.0 ± 3.5  1.4
 50 90.3 ± 8.6  1.0  92.3 ± 7.4  1.0  9.7 ± 5.0  1.5  9.0 ± 2.6  1.1
 167 85.3 ± 22.6  0.9  69.3 ± 17.8  0.7 7.0 ± 3.6  1.1  11.7 ± 5.0  1.5
 500 75.7 ± 9.1 P  0.8  75.7 ± 7.1 P  0.8  7.3 ± 2.5 P  1.1  11.3 ± 4.0 P  1.4
 1667  95.3 ± 21.4 P  1.0  76.0 ± 7.2 P  0.8  6.7 ± 2.5 P  1.0  8.7 ± 2.1 P  1.1
 5000  87.0 ± 10.8 P  0.9  83.7 ± 17.5 P  0.9  9.0 ± 6.2 P  1.3  9.0 ± 5.3 P  1.1
 Positive control  759.7 ± 24.8  8.2 550.7 ± 66.0  5.9  174.7 ± 21.4  26.2  685.7 ± 60.1  85.7

P = Precipitate

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 revertants per plate± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio
Acetone   13.7 ± 4.0  -  11.0 ± 5.6  -  8.3 ± 2.1  -  12.7 ± 2.5  -  21.7 ± 1.5  -  32.0 ± 4.4  -
 17  10.0 ± 3.6  0.7  12.3 ± 2.5  1.1  9.3 ± 2.5  1.1  14.0 ± 3.6  1.1  21.7 ± 11.0  1.0  28.0 ± 4.6  0.9
 50  13.0 ± 3.0  1.0  16.7 ± 7.5  1.5  8.0 ± 3.0  1.0  13.0 ± 3.6  1.0  17.3 ± 1.2  0.8  29.0 ± 8.2  0.9
 167  13.0 ± 2.0  1.0  10.3 ± 5.0  0.9  6.0 ± 0.0  0.7  14.0 ± 2.6  1.1  25.3 ± 3.8  1.2  32.0 ± 4.4  1.0
 500  14.7 ± 2.3 P  1.1  5.3 ± 4.0 P  0.5  10.0 ± 3.5 P  1.2  14.0 ± 6.6 P  1.1  21.7 ± 5.7 P  1.0  28.7 ± 2.5 P  0.9
 1667  11.0 ± 1.7 P  0.8  10.0 ± 3.5 P  0.9  9.7 ± 4.7 P  1.2  15.7 ± 3.1 P  1.2  20.3 ± 4.6 P  0.9  31.3 ± 9.1 P  1.0
 5000  14.3 ± 4.0 P  1.0  13.7 ± 4.5 P  1.2  10.3 ± 0.6 P  1.2  13.0 ± 4.6 P  1.0  23.3 ± 4.0 P  1.1  26.3 ± 4.0 P  0.8
 Positive Control  614.7 ± 68.4  45.0  306.7 ± 26.3  27.9 2165.7 ± 699.6 259.9  159.7 ± 37.3  12.6  752.7 ± 14.5  34.7  270.3 ± 24.8  8.4

P = Precipitate

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 ± 8.0  -  100.0 ± 13.5  -  8.0 ± 2.6 - 7.0 ± 1.7  -
 17  92.7 ± 10.1  0.9  94.3 ± 6.4  0.8  8.3 ± 3.8  1.0  8.7 ± 4.2  1.2
 50  86.7 ± 11.6  0.9  104.0 ± 4.0  1.0  7.7 ± 4.1  1.0  12.0 ± 2.6  1.7
 167  84.0 ± 5.6  0.8  87.0 ± 3.5  1.0  4.0 ± 2.0  0.5  9.3 ± 2.1  1.3
 500  93.0 ± 4.0 P  0.9  96.3 ± 19.1 P  0.9  6.7 ± 2.1 P  0.8  8.7 ± 3.2 P  1.2
 1667  84.3 ± 12.9 P  0.8  85.7 ± 13.5 P  0.9  10.3 ± 5.0 P  1.3  11.3 ± 3.2 P  1.6
 5000  99.7 ± 16.4 P  1.0  93.7 ± 6.4 P  0.9  7.7 ± 3.1 P  1.0  9.3 ± 3.2 P  1.3
 Positive control  1191.7 ± 75.8  11.8  775.0 ± 29.8  7.8  184.0 ± 16.1  23.0  669.3 ± 40.3  95.6

P = Precipitate

Conclusions:
Interpretation of results (migrated information):
negative

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

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, alkenes 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 test 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. No toxicity to the bacteria was observed in either the absence or the presences of S9 mix. Precipitation of HF-1000 occurred at 500 µg per plate and above in both assays, in both absence and presence of S9 mix.

It was concluded that Hydrocarbons, C12-C16, n-alkanes, isoalkanes, alkenes 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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Metabolic activation system:
S9 enzymes from the livers of Aroclor 1254-treated adult, male Fisher rats
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
other: not tested
Positive controls validity:
valid
Key result
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 500, 1667 and 5000 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 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 (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'.

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  87  -  95  -
17  62  0.7  87  0.9
 50  81  0.9  112  1.2
 167  78  0.9  81  0.9
 500  79 P  0.9  79 P  0.8
 1667  75 P  0.9  112 P  1.2
 5000  75 P  0.9  66 P  0.7

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
Acetone 11.3 ± 2.1  -  13.7 ± 7.8  -  9.7 ± 3.1  -  7.3 ± 3.2  -  17.0 ± 5.6  -  33.0 ± 10.4  -
 17  7.7 ± 4.9  0.7  11.0 ± 3.5  0.8  10.7 ± 1.2  1.1  13.0 ± 3.6  1.8  18.3 ± 3.5  1.1  34.0 ± 7.0 1.0
 50  14.3 ± 2.9  1.3  8.7 ± 2.9  0.6  12.0 ± 1.0  1.2  8.0 ± 4.6  1.1  20.7 ± 5.5  1.2  31.3 ± 2.1  0.9
 167  8.7 ± 2.9  0.8  11.0 ± 3.5  0.8  7.7 ± 4.6  0.8  13.0 ± 1.7  1.8  22.7 ± 7.6  1.3  29.3 ± 4.2  0.9
 500  10.0 ± 6.6 P  0.9  11.7 ± 3.8 P  0.9  6.7 ± 2.5 P  0.7  12.7 ± 2.1 P  1.7  19.3 ± 6.4 P  1.1  26.7 ± 5.9 P  0.8
 1667  6.3 ± 5.1 P  0.6  11.0 ± 4.6 P  0.8  7.3 ± 2.3 P  0.8  14.0 ± 4.6 P  1.9  24.0 ± 8.5 P  1.4  27.3 ± 5.1 P  0.8
 5000  8.3 ± 2.9 P  0.7  14.0 ± 4.6 P  1.0  12.7 ± 3.1 P  1.3  13.3 ± 3.2 P  1.8  23.7 ± 8.3 P  1.4  30.3 ± 2.3 P  0.9
 Positive Control  444.0 ± 49.0  39.2  331.0 ± 26.5  24.2  3764.0 ± 423.2  389.4  272.0 ± 14.1  37.1  918.7 ± 184.7 54.0   365.0 ± 12.8  11.1

P = Precipitate

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 92.7 ± 5.5  -  93.0 ± 18.5  -  6.7 ± 4.5  -  8.0 ± 3.5  -
 17  91.3 ± 11.4  1.0  82.3 ± 13.6  0.9  7.7 ± 4.7  1.1  11.0 ± 3.5  1.4
 50 90.3 ± 8.6  1.0  92.3 ± 7.4  1.0  9.7 ± 5.0  1.5  9.0 ± 2.6  1.1
 167 85.3 ± 22.6  0.9  69.3 ± 17.8  0.7 7.0 ± 3.6  1.1  11.7 ± 5.0  1.5
 500 75.7 ± 9.1 P  0.8  75.7 ± 7.1 P  0.8  7.3 ± 2.5 P  1.1  11.3 ± 4.0 P  1.4
 1667  95.3 ± 21.4 P  1.0  76.0 ± 7.2 P  0.8  6.7 ± 2.5 P  1.0  8.7 ± 2.1 P  1.1
 5000  87.0 ± 10.8 P  0.9  83.7 ± 17.5 P  0.9  9.0 ± 6.2 P  1.3  9.0 ± 5.3 P  1.1
 Positive control  759.7 ± 24.8  8.2 550.7 ± 66.0  5.9  174.7 ± 21.4  26.2  685.7 ± 60.1  85.7

P = Precipitate

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 revertants per plate± SD  Ratio  Mean revertants per plate ± SD  Ratio  Mean revertants per plate ± SD  Ratio
Acetone   13.7 ± 4.0  -  11.0 ± 5.6  -  8.3 ± 2.1  -  12.7 ± 2.5  -  21.7 ± 1.5  -  32.0 ± 4.4  -
 17  10.0 ± 3.6  0.7  12.3 ± 2.5  1.1  9.3 ± 2.5  1.1  14.0 ± 3.6  1.1  21.7 ± 11.0  1.0  28.0 ± 4.6  0.9
 50  13.0 ± 3.0  1.0  16.7 ± 7.5  1.5  8.0 ± 3.0  1.0  13.0 ± 3.6  1.0  17.3 ± 1.2  0.8  29.0 ± 8.2  0.9
 167  13.0 ± 2.0  1.0  10.3 ± 5.0  0.9  6.0 ± 0.0  0.7  14.0 ± 2.6  1.1  25.3 ± 3.8  1.2  32.0 ± 4.4  1.0
 500  14.7 ± 2.3 P  1.1  5.3 ± 4.0 P  0.5  10.0 ± 3.5 P  1.2  14.0 ± 6.6 P  1.1  21.7 ± 5.7 P  1.0  28.7 ± 2.5 P  0.9
 1667  11.0 ± 1.7 P  0.8  10.0 ± 3.5 P  0.9  9.7 ± 4.7 P  1.2  15.7 ± 3.1 P  1.2  20.3 ± 4.6 P  0.9  31.3 ± 9.1 P  1.0
 5000  14.3 ± 4.0 P  1.0  13.7 ± 4.5 P  1.2  10.3 ± 0.6 P  1.2  13.0 ± 4.6 P  1.0  23.3 ± 4.0 P  1.1  26.3 ± 4.0 P  0.8
 Positive Control  614.7 ± 68.4  45.0  306.7 ± 26.3  27.9 2165.7 ± 699.6 259.9  159.7 ± 37.3  12.6  752.7 ± 14.5  34.7  270.3 ± 24.8  8.4

P = Precipitate

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 ± 8.0  -  100.0 ± 13.5  -  8.0 ± 2.6 - 7.0 ± 1.7  -
 17  92.7 ± 10.1  0.9  94.3 ± 6.4  0.8  8.3 ± 3.8  1.0  8.7 ± 4.2  1.2
 50  86.7 ± 11.6  0.9  104.0 ± 4.0  1.0  7.7 ± 4.1  1.0  12.0 ± 2.6  1.7
 167  84.0 ± 5.6  0.8  87.0 ± 3.5  1.0  4.0 ± 2.0  0.5  9.3 ± 2.1  1.3
 500  93.0 ± 4.0 P  0.9  96.3 ± 19.1 P  0.9  6.7 ± 2.1 P  0.8  8.7 ± 3.2 P  1.2
 1667  84.3 ± 12.9 P  0.8  85.7 ± 13.5 P  0.9  10.3 ± 5.0 P  1.3  11.3 ± 3.2 P  1.6
 5000  99.7 ± 16.4 P  1.0  93.7 ± 6.4 P  0.9  7.7 ± 3.1 P  1.0  9.3 ± 3.2 P  1.3
 Positive control  1191.7 ± 75.8  11.8  775.0 ± 29.8  7.8  184.0 ± 16.1  23.0  669.3 ± 40.3  95.6

P = Precipitate

Conclusions:
Interpretation of results (migrated information):
negative

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

The source substance, Hydrocarbons, C12-C16, n-alkanes, isoalkanes, alkenes 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 test 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. No toxicity to the bacteria was observed in either the absence or the presences of S9 mix. Precipitation of HF-1000 occurred at 500 µg per plate and above in both assays, in both absence and presence of S9 mix.

It was concluded that the source substance, Hydrocarbons, C12-C16, n-alkanes, isoalkanes, alkenes, 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.

Genetic toxicity in vivo

Description of key information

No evidence of mutagenic activity was seen in this in vivo test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material: Alcohols, C2-33, manuf. of, by-products from overheads
- Substance type: Product, HF-1000
- Physical state: clear yellow liquid
- Odour: oily/solvent
- Purity test date: 2010/10/24
- Lot/batch No.: 68310
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males: 217-265 g; females: 146-184 g
- Assigned to test groups randomly: each animal was assigned a unique coded number from a computer numbered sequence ranging from 201 to 243
- Fasting period before study: no
- Housing: 5 animals per cage, gender separated, polycarbonate/stainless steel fgrid tops (61x43.5x24 cm)
- Diet : Food was freely available to the rats all times; International certified rodent chow supplied by IPS Ltd., UK
- Water: Tap water (ad libitum)
- Acclimation period: not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9-22.5
- Humidity (%): 47-79
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% w/v carboxymethyl cellulose with 0.1% w/v Tween 80 in water
- Justification for choice of solvent/vehicle: not mentioned
Details on exposure:
All animals were exposed to test or control materials via the intraperitoneal dose route.
Frequency of treatment:
single application
Post exposure period:
24 hours
Remarks:
Doses / Concentrations:
10 ml/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Vehicle: 5m + 5f
100 mg/kg bw: 5m
200 mg/kg bw: 5m
400 mg/kg bw: 10 m + 10 f
positive control: 3m
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal
- Dose: 50 mg/kg bw
- Vehicle: see description
- Total application volume: 10 ml/kg bw
- post exposure period: 24 hours
Tissues and cell types examined:
bone marrow; polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: preliminary range finding test were undertaken prior to the micronucleus test.
Range finding study: 400, 600, 800 and 1000 mg/kg (for each doses 1m+1f)
The rats were observed for clinical signs or motality.

DETAILS OF SLIDE PREPARATION:
Rats were killed by CO2 asphyxiation. One femur of each rat was promptly removed and freed adherent tissue. A small hole was made in the neck of one femur and the bone marrow flushed with.The tubes were centrifuged to pellet the cells. All but a few drops of supernatant fluid were discarded.
Two slides were prepared from each tube per animal. The smears were left to air-dry. They were then fixed in methanol and immeresd in Giemsa stain solution.

METHOD OF ANALYSIS:
One of the two prepared slides was selected for examination. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-NCE).
Evaluation criteria:
The mean micronucleus incidence in vehicle control-dosed and untreated CD rats has, in this laboratory, been determined as 0.04 ± 0.05%: a range of 0.01-0.13% per group of 5-7 rats and 0.02-0.11% per group of 10-12 rats. This frequency is an agreement with published data for miccronucleus tests with CD rats (Tamura et al, 1990; Salmone and Mavourin, 1994). These historical data have been used in the evaluation of the response in this test.
Statistics:
No statistical analysis was performed as the levels of MN-PCE induction fell within the determined historical control frequencies.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:

200, 400 and 600 mg/kg bw no deaths; 1000 mg/kg two deaths

- Clinical signs of toxicity in test animals:
Range-finding study:Clinical signs affecting the rats behaviour, breathing and posture were observed in the range finding tests. Based on these toxicity observations, the maximum tolerated dose of HF-1000 was judged to be in the region of 400 mg/kg bw.

Test itemgroup: There was no indication of bone marrow toxicity in any of the test item dose groups.
RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
No animal deaths occured in the main micronucleus test. No animals displayed signs of abnormality.

- Induction of micronuclei:
Positive control: Exposure of rats to the positive control agent, cyclophosphamide, induced large increases in bone marrow micronuclei. The mean MN-PCE frequency for the rats was 2.68%. An evident increase in the number of MN-NCE was also observed.
Test substance: There was no indication that the test substance induced bone marrow micronuclei in the treated rats. The highest MN-PCE frequency recorded for the test item was in the high dose females, where an incidence of 0.03% was observed.
Treatment Dose (h) Sex No. of rats scored Erythrocytes
NCE PCE PCE/NCE Mean ± SD
No of MN-NCE PCE analysed No of MN-PCE MN-PCE [%]
Vehicle 0 + 24 m 5 2 10006 3 0.03 0.68 ± 0.16
f 5 2 10001 3 0.03 0.72 ± 0.10
m+f 10 4 20007 6 0.03 0.70 ± 0.13
100 mg/kg 0 + 24 m 5 0 10000 2 0.02 0.60 ± 0.07
200 mg/kg 0 + 24 m 5 0 10002 2 0.02 0.65 ± 0.14
400 mg/kg 0 + 24 h m 10 1 20002 2 0.01 0.66 ± 0.14
f 10 8 20002 7 0.03 0.61 ± 0.11
m+f 20 9 40004 9 0.02 0.63 ± 0.11
positive control 0 + 24 h m 3 89 α 6001 161 2.68 φ 0.31 ± 0.01

α = Evident response in NCE

φ = Positive response in PCE

Conclusions:
Interpretation of results (migrated information): negative
It was conducted that Hydrocarbons, C12-C16, n-alkanes, isoalkanes, alkenes did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 400 mg/kg bw in male and female CD rats using 0h + 24 h intraperitoneal and 48 sampling regimen.

Additional information

The negative results obtained for the source substance using in vitro and in vivo genotoxicity assays do not warrant the classification of the registered substance as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.


Short description of key information:
The source substance gave negative results in the genetic toxicity tests listed below
Genetic Toxicity in vitro – Bacterial reverse mutation assay (OECD TG 471)

Genetic Toxicity in vitro- Mammalian mtagenicity assay (OECD TG 476)
Genetic Toxicity in vivo – Micronucleus Assay in Mouse Bone Marrow (OECD TG 474)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The negative results obtained for the source substance using in vitro and in vivo genotoxicity assays do not warrant the classification of the registered substance as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.