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Diss Factsheets

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
other: EC 0 J. No. L 136 (2000)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6,8-trimethylnonan-4-ol
EC Number:
204-606-8
EC Name:
2,6,8-trimethylnonan-4-ol
Cas Number:
123-17-1
Molecular formula:
C12H26O
IUPAC Name:
2,6,8-trimethylnonan-4-ol
Details on test material:
- Name of test material (as cited in study report): 2,6,8-trimethyl-4-nonanol
- Molecular weight (if other than submission substance): 186.3
- Physical state: Liquid
- Analytical purity: The purity of the test material sample was determined to be 96.05% trimethylnonanol, with five minor components present at 0.1% or greater by gas chromatography. Identity was confirmed by infrared (IR) spectroscopy and gas chromatography-mass spectrometry (GC/MS).

Method

Target gene:
histidine (his+) or tryptophan (trp+)
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:
The S-9 activation system consists of an NADPH regeneration system plus the S-9 fraction of rat liver homogenate. The S-9 fraction of rat liver homogenates was prepared from Aroclor 1254 induced male Sprague-Dawley rats.
Test concentrations with justification for top dose:
The concentrations selected under these conditions were 5000, 3333, 1000,333, 100,33, 10,3.3, and 1 ug/plate in the presence and absence of S-9.
Vehicle / solvent:
2,6,8-trimethyl-4-nonano(TMN) was dissolved in DMSO. The selected concentrations of the test material in the treatment medium or in the stock solutions used for the treatment in the main assay were verified by gas chromatography with flame ionization detection (GC/FID).
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: See Table below
Details on test system and experimental conditions:
Preliminary Assay
Selection of dose levels for the main mutagenicity assay was based upon the toxicity and precipitation profile of the test article assessed in a preliminary assay. The tester strains were exposed to the test article via the pre-incubation modification of the Ames test described by Maron and Ames (1 983). In the preincubation methodology the tester strain and the test article are pre-incubated at 37C for approximately 20 minutes prior to the addition of molten agar. The test material was evaluated up to a concentration of 5000 ug/plate. The concentrations selected under these conditions were 5000, 3333, 1000,333, 100,33, 10,3.3, and 1 ug/plate in the presence and absence of S-9.

For the assay with activation, the bacteria (0.1 ml), test article (0.05 ml of the appropriately diluted test material or solvent), and the S-9 mix (0.5 ml) were placed into sterile tubes incubated at 37OC for approximately 20 minutes and 2 ml of top agar (supplemented with trace amounts of histidine and biotin or tryptophan) was added, mixed and poured onto minimal glucose agar plates. With some solvents such as dimethyl sulfoxide and water, the volume of the test materiaVsolverf may have been increased up to 0.1 ml in order to achieve the desired concentrations for the testing of insoluble materials. For the non-activation assay, S-9 mix was omitted and replaced by 0.5 ml of 0.2M phosphate buffer, pH 7.4. The plates were then incubated for approximately 52 f 4 hour in an incubator at 37C. This preliminary assay was conducted by exposing all strains to negative controls (three plates) and positive controls (three plates) and nine concentrations of test article (three platesldose), in both the presence and absence of S-9 activation. The highest concentration evaluated represented the limit concentration for this assay.

Mutagenicity Assay
In selecting concentration levels for the mutagenicity assay, the following guidelines were employed. Concentrations were selected based on the preliminary assay such that precipitate did not interfere with the counting of colonies. Whenever possible, the highest concentration for the mutagenicity assay was selected to give some indication of toxicity without exceeding 5 mg/plate. For freely soluble, nontoxic test articles, the highest concentration level was 5 mg/plate. For precipitating, nontoxic test articles, the highest concentration level was selected in an attempt to yield precipitate at only the top one or two concentration levels.

The test article was tested at a minimum of 7 concentration levels along with appropriate negative and positive controls with all tester strains (TA98, TA100, TA1535, TA1537, and WP2uvrA), with and without S-9 activation. All concentration levels of the test article, negative controls, and positive controls were plated in triplicate. A confirmatory assay was performed in the event of equivocal or positive findings in the mutagenicity assay.
Evaluation criteria:
Plate Counts/Evaluation
Prior to scoring the assay plates, all tester strain cultures were checked for the appropriate genetic markers. The condition of the bacterial background lawn was evaluated for evidence of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control and recorded along with the revertant counts for that concentration. Revertant colonies were counted either by automated colony counter or
manually (generally when fewer than 100 colonies). Plates tht were not evaluated immediately following the incubation period were held at 5 +/- 3OC, until the colony counting and bacterial background lawn evaluation could take place but not more than two weeks.

Criteria For Determination of a Valid Test
The following criteria must have been met for the mutagenicity assay to be considered valid.

Tester Strain Integrity
To demonstrate the presence of the rfa mutation, all S. typhimurium tester strain cultures must have exhibited sensitivity to crystal violet. To demonstrate the presence of the pKM101 plasmid R-factor, tester strain cultures of TA98 and TA100 must have exhibited resistance to ampicillin as described above.

Negative Control Values
Based on in house and literature derived historical control data, all tester strain cultures should have exhibited characteristic numbers of revertants per plate in the negative controls (vehicle). The mean revertants per plate should have been within the following ranges (inclusive): TA98, 10-50; TA100, 80-200; TA1535,5-45; TA1537, 3-21; WP2uvrA, 10-54. Untreated controls, when part of the design, must have also been within the ranges cited above.

Positive Control Values
Each mean positive control value must have exhibited at least a 3.0- fold increase over the respective mean negative control value (vehicle) for each tester strain.

Toxicity (see below)
Statistics:
Means and Standard Deviations were calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
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:
In the presence of S-9, TA98 and TA1537 exhibited cytotoxicity at >/-1000 ug/plate and TA100 from >/-100 ug/plate. In the absence of S-9 TA100 and TA1537 exhibited cytotoxicity from 10 ug/plate and TA98 from 33 ug/plate. Others tested up to limit conc.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The mutagenicity of 2,6,8-trimethyl-4-nonano(lt rimethylnonanol) was evaluated using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and E. coli WP2uvrA in the presence and absence of a metabolic activation system (S-9). Based on its limited solubility in water, DMSO was selected as the vehicle due to its solubility in this solvent and its compatibility with the test system.

Preliminary Assay
A preliminary assay was conducted on all tester strains (3 plates per treatment) in both the presence and absence of metabolic activation (S-9 mix) at concentrations ranging from 1-5000 ug/plate. In the absence of S-9, TA98 exhibited cytotoxicity from 100 ug/plate while TA100 and TA1537 exhibited cytoxicity from 10 and 33 ug/plate, respectively. In the presence of S-9 TA98 and TA1537 exhibited cytotoxicity at 333 ug/plate and above, while TA100 exhibited cytotoxicity from 100 ug/plate. TA1535 and WP2uvrA exhibited no cytotoxicity in either the absence or presence of metabolic activation. Mean revertants for the negative and positive controls for all strains exhibited numbers that satisfied the criteria for a valid assay. No significant increases in mutant counts were observed that satisfied the criteria (TA98, TA1535, TA1537, and WP2uvrA: 3-fold and TA100: 2-fold), for a positive assay response. No precipitation was observed on any of the plates at any concentration in either the presence or absence of S-9.

Mutagenicity Assay
The mutagenicity assay was conducted up to a maximal level of 5000 ug/plate in all tester strains in the presence of metabolic activation and for TA98, TA1535, and E.coli WP2uvrA in the absence of S-9. TA100, and TA1537 were evaluated from 0.1-333 ug/plate in the absence of S-9. In the presence of S-9, TA98 and TA1537 exhibited cytotoxicity at 1000 and 5000 ug/plate and TA100 from 100-5000 ug/plate. In the absence of S-9 TA100 and TA1537 exhibited cytotoxicity from 10 ug/plate and TA98 from 33 ug/plate. The other tester strains (TA1535 and WP2uvrA) did not exhibit toxicity in terms of decreased background lawn up to the maximal concentration evauluated (5000 ug/plate) in either the presence or absence of metabolic activation. No precipitation was observed on any of the plates at any concentration in either the presence or absence of S-9. Theconcentrations of test material in the dosing solutions were analytically confirmed to be between 92- 1 15% of the stated values. The positive and negative control revertant counts satisfied the criteria for a valid assay. Again, no significant increases in mutant counts were observed that satisfied the criteria for a positive assay response.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table. Mutagenicity Assay Results (Assay B1)

               Mean + SD
 ug/Plate  TA98  TA100  TA1535  TA1537  WP2uvrA
 With S-9          
 0.0  32 + 8.1  136 + 19.1  15 + 4.7  11 + 4.0  41 + 12.0
 1.0  33 + 3.0  143 + 17.2  18 + 4.0  10 + 3.5  47 + 7.0
 3.3  26 + 9.5  158 + 13.7  18 + 5.1  11 + 3.5  46 + 5.1
 10  32 + 5.5  139 + 18.6  15 + 2.3  9 + 2.1  47 + 9.0
 33  30 + 4.4  153 + 6.8  14 + 1.5  11 + 4.0   45 + 13.0
 100  30 + 1.0  69 + 16.81  14 + 2.6  9 + 2.1  47 + 6.5
 1000  31 + 8.01  29 + 11.42  14 + 2.6  0 + 0.03  36 + 11.2
 5000  21 + 4.61  13 + 4.02  12 + 4.7  0 + 0.03  43 + 1.5
 PCb  287 + 25.4  1310 + 34.4  120 + 10.1  127 + 8.1  421 + 26.1
 Without S-9          
 0.0  25 + 4.9  123 + 28.9  19 + 4.2  8 + 2.1  39 + 4.6
 0.100  ---  124 + 6.5  ---  9 + 6.4  ---
 0.333  ---  129 + 17.8  ---  11 + 2.9  ---
 1.0  23 + 4.4  128 + 14.6  21 + 2.1  7 + 2.6  38 + 2.5
 3.3  25 + 7.5  122 + 4.4  18 + 2.1  12 + 2.1  35 + 7.8
 10  22 + 3.6  67 + 9.51  18 + 2.5  4 + 1.51  41 + 1.0
 33  23 + 2.01  58 + 25.01/2  13 + 2.5  3 + 3.11/3  36 + 4.0
 100  24 + 3.81  40 + 52.91/2/3  16 + 5.8  2 + 0.6 1/3  44 + 4.9
 333  ---  0 + 0.03  ---  0 + 0.03  ---
 1000  17 + 7.51  ---  15 + 6.4  ---  36 + 7.6
 5000  19 + 2.61  ---  10 + 2.6  ---  40 + 2.6
 PCc  660 + 52.5  1453 + 28.9  1465 + 29.1  845 + 45.8  384 + 40.5

1 = Slightly reduced, 2= Moderately reduced, 3 = Severely reduced or absent

b TA98: Benzo(a)pyrene. 2.5 ug/plate

TA100: 2-Aminoanthracene. 2.5 ug/plate

TA1535: 2-Aminoanthracene, 2.5 ug/plate

TA1537: 2-Aminoanthracene, 2.5 ug/plate

WP2uvrA: 2-Aminoanthracene, 25.0 ug/plate

cTA98: 2-Nitrofluorene. 5.0 ug/plate

TA100: Sodium azide, 10.0 ug/plate

TA1535: Sodium azide. 10.0 ug/plate

TA1537: ICR-191, 2.0 ug/plate

WP2uvrA: 4-Nitroquinoline-N-oxide, 0.4 ug/plate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of the study, trimethylnonanol was concluded to be negative in the bacterial reverse mutation assay under the conditions used in the study.
Executive summary:

The mutagenicity of 2,6,8-trimethyl-4- nonanol (trimethylnonanol) was evaluated using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and E.coli WP2uvrA in the presence and absence of a metabolic activation system (S-9). Dimethylsulfoxide (DMSO) was selected as the solvent of choice due to the solubility of the test material in this solvent and its compatibility with the test system. A preliminary assay was conducted on all tester strains in both the presence and absence of metabolic activation at nine concentrations ranging from 1-5000 μg/plate. In the absence of S-9, TA98 exhibited cytotoxicity from 100 μg/plate and TAl00 and TA1537 from 33 μg/plate. In the presence of S-9, TA98 and TA1537 exhibited cytotoxicity at 333 μg/plate and above and TAl00 from 100 μg/plate. TA1535 and WP2uvrA exhibited no cytotoxicity in either the absence or presence of metabolic activation. Mean revertants for the negative and positive controls for all strains exhibited numbers that satisfied the criteria for a valid assay. No significant increases in mutant counts were observed that satisfied the criteria (TA98, TA1535, TA1537, and WP2uvrA: 3-fold and TA100: 2-fold), for a positive assay response. No precipitation was observed on any of the plates at any concentration in either the presence or absence of S-9.

The mutagenicity assay was conducted at 7-8 concentrations up to a maximal level of 5000 μg/plate in all tester strains in the presence and absence of metabolic activation. In the presence of S-9, TA98 and TA1537 exhibited cytotoxicity at 1000 and 5000 μg/plate and TA100 from 100-5000 μg/plate. In the absence of S-9, TA100 and TA1537 exhibited cytotoxicity from 10 μg/plate and TA98 from 33 μg/plate. The other tester strains (TA1535 and WP2uvrA) did not exhibit toxicity in terms of decreased background lawn up to the maximal concentration evaluated (5000 μg/plate) in either the presence or absence of metabolic activation. The concentrations of test

material in the dosing solutions were analytically confirmed to be between 92- 115% of the stated values. The positive and negative control revertant counts satisfied the criteria for a valid assay. Again, no significant increases in mutant counts were observed that satisfied the criteria for a positive assay response. Hence, trimethylnonanol is concluded to be negative in the bacterial reverse mutation assay under the conditions used in this study.