Reaction mass of 2,6-dimethylheptan-4-ol and 4,6-dimethylheptan-2-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: other: reverse gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Not Applicable

GLP compliance:

yes

Type of assay:

other: To evaluate the ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2,6-dimethylheptan-4-ol and 4,6-dimethylheptan-2-ol

EC Number:

939-420-2

Molecular formula:

C9H20O

IUPAC Name:

Reaction mass of 2,6-dimethylheptan-4-ol and 4,6-dimethylheptan-2-ol

Details on test material:

Sponsor’s Identification: Diisobutyl Carbinol
Lot No. 7897
Sample Record No. 90008
Date Received: January 29, 2002
Physical Description: Transparent, colorless liquid
Storage Conditions: Ambient temperature
Molecular Formula: C9H20O
Molecular Weight: 144.2
Purity/Characterization: 98.34 % (sum of both isomers) as determined by gas chromatography area percent, percent water (Karl Fischer
coulometric titration). Identification: infared spectroscopy (FTIR) and gas chromatographic mass spectroscopy (GC/MS).
CAS Numbers: 108-82-7 (2,6-Dimethyl-4-heptanol)
51079-52-8 (4,6-Dimethyl-2-heptanol)

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The Salmonella typhimurium tester strains in use at Covance were received directly from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley. The Escherichia coli tester strain, WP2uvrA, was received from The National Collection of Industrial Bacteria, Torrey Research
Station, Scotland (United Kingdom).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Metabolic Activation System

Test concentrations with justification for top dose:

0.00, 6.67, 10., 33.3, 66.7, 100, 333, 337, 1000, 3330, 5000 μg DIBC/Plate

Vehicle / solvent:

Vehicle: dimethylsulfoxide (DMSO)
Justification of vehicle: The test article was observed to be insoluble in water at concentrations of approximately 100, 50.1, and 25.0 mg per mL. The test article was observed to form a solution in dimethylsulfoxide (DMSO) at a concentration of 99.7 mg per mL. For this reason, DMSO
(CAS# 67-68-5, Acros Organics, Lot Nos. A013154701 and A014587401) was selected as the vehicle for this study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

in the presense and absense of S-9 mix

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 2-nitrofluorene, 2-aminoanthracene, sodium azide, ICR-191, 4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

Rangefinding Assay
The growth inhibitory effect (cytotoxicity) of the test article to the test system was determined inorder to allow the selection of appropriate
concentrations to be tested in the mutagenicity assay.

Design.
The rangefinding study was performed using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix. Ten concentrations of test article were tested at one plate per concentration. The test article was checked for cytotoxicity up to a maximum concentrationof 5000 μg per
plate.

Rationale.
The cytotoxicity of the test article observed on tester strain TA100 is generallyrepresentative of that observed on the other Salmonella typhimurium tester strains and because of the comparatively high number of spontaneous revertants per plate observed with this strain, gradations of
cytotoxicity can be readily discerned from routine experimental variation. TheEscherichia coli tester strain WP2uvrA does not possess the rfa wall
mutation that the Salmonella typhimurium strains have and thus, a different range of cytotoxicity may be observed. Also, the cytotoxicity induced by a test article in the presence of S9 mix may vary greatly from that observed in the absence of S9 mix. Therefore, this would require that different
test article concentration ranges be tested in the mutagenicity assay based on the presence or absence of the microsomal enzymes.

Evaluation of the Rangefinding Assay.
Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial
background lawn. Selection of the Maximum Concentration for the Mutagenicity Assay. Cytotoxicity was observed in the rangefinding study, and
the highest concentration of test article used in the subsequent mutagenicity assay was a concentration which gave a reduction of revertants per
plate and/or a thinning or disappearance of the bacterial background lawn.

Mutagenicity Assay
Design. The assay was performed using tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA both in the presence and absence of S9 mix
along with the appropriate vehicle and positive controls. The concentrations of test article were selected based on the results of the rangefinding
assay. The results of the initial mutagenicity assay were confirmed in an independent experiment.

Frequency and Route of Administration.
The tester strains were exposed to the test article via the preincubation modification of the Ames Test originally described by Yahagi et al. (1975) and
Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the preincubation
methodology, S9 mix (or phosphate buffer, whereappropriate), the tester strain, and the test article were preincubated prior to the addition of
molten agar. The agar and the preincubation reaction mixture were mixed and then overlaid onto a minimal agar plate. Following incubation,
revertant colonies were counted. All concentrations of the test article, the vehicle controls and the positive controls were plated in triplicate (see
Protocol Deviation, page 18).

Plating Procedures
These procedures were used in both the rangefinding study and the mutagenicity assay. Eachplate was labeled with a code which identified the test
article, test phase, tester strain, activation condition and concentration level. The S9 mix and dilutions of the test article were prepared immediately
prior to their use. When S9 mix was required, 500 μL of S9 mix was added to 13 x 100 mm glass culture tubes, which had been pre-heated to
37 ± 2°C. To these tubes was added 100 μL of tester strain and 50 μL of vehicle or test article concentration. When S9 mix was not required, 500 μL of 0.1M phosphate buffer was substituted for the S9 mix. After the required components had been added, the mixture was vortexed and allowed to
incubate for 20 ± 2 minutes at 37 ± 2°C. Two mL of molten selective top agar was then added to each tube, and the mixture was vortexed and
overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay solidified, the plates were
inverted and incubated for 52 ± 4 hours at 37 ± 2°C. Positive control articles were plated using a 50 μL plating aliquot.

Scoring the Plates
Plates which were not evaluated immediately following the incubation period were held at >0°C to 10°C until such time that colony counting and
bacterial background lawn evaluation could take place. Bacterial Background Lawn Evaluation. The condition of the bacterial background lawn was
evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate.
Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that
concentration level. Lawns were scored as normal (N), reduced (R), obscured by precipitate (O), macroscopic precipitate present (P), absent (A), or
enhanced (E); contaminated plates (C) also were noted.

Counting Revertant Colonies.
Revertant colonies were counted either by automated colony
counter or by hand.

Evaluation criteria:

Assay Acceptance Criteria
Before assay data were evaluated, the criteria for a valid assay had to be met. The following criteria were used to determine a valid assay: rfa Wall
Mutation that Salmonella strain exhibited sensitivity to crystal violet. Demonstrate the presence ofpKM101 plasmid in TA98 and TA100 strains.
Number of Spontaneous Revertants in vehicle controls (TA98 8-60, TA100 60-240, TA1535 4-25, TA1537 2-25, WP2uvrA 5-40). Demonstrate that Strain Culture Density was

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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:

not examined

Positive controls validity:

valid

Additional information on results:

Rangefinding Assay
Concentrations tested in the mutagenicity assay were selected based on the results of the rangefinding assay conducted on the test article using
tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per concentration. Ten concentrations of test
article, from 6.67 to 5000 μg per plate, were tested and the results are presented in Data Tables 1 and 2. These data were generated in Experiment
23500-A1. Cytotoxicity was observed at concentrations ≥333 μg per plate with both tester strains in the presence or absence of S9 mix as
evidenced by a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn. No
test article precipitate was observed on any of the plates in the presence or absence of S9 mix. See Tables 1 and 2 in Results below.

Mutagenicity Assay
The preincubation mutagenicity assay results for Diisobutyl Carbinol are presented in Data Tables 3 through 7. These data were generated in
Experiments 23500-B1 and 23500-C1. The data are presented as individual plate counts (Tables 3 and 5) and as mean revertants per plate ±
standard deviation (Tables 4 and 6) for each treatment and control group. The tester strains used in the preincubation mutagenicity assay were
Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted
with six concentrations of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three
plates per concentration. Concentrations tested in the initial mutagenicity assay were selected based on the results of the rangefinding study. The
concentrations tested in the initial mutagenicity assay with all tester strains in the presence of S9 mix were 3.33, 10.0, 33.3, 100, 333, and 1000 μg
per plate. The concentrations tested in the initial mutagenicity assay with all tester strains in the absence of S9 mix were 1.00, 3.33, 10.0, 33.3, 100, and 500 μg per plate. In the initial mutagenicity assay (Experiment 23500-B1, Tables 3 and 4), all data were acceptable, and no positive increases in
the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. Concentrations
tested in the confirmatory assay were selected, in conjunction with the Sponsor, based on the results of the initial mutagenicity assay. The
concentrations tested in the confirmatory mutagenicity assay with all tester strains in the presence of S9 mix were 3.33, 10.0, 33.3, 100, 333, and
1000 μg per plate. The concentrations tested in the confirmatory mutagenicity assay with all tester strains in the absence of S9 mix were 1.00, 3.33, 10.0, 33.3, 100, and 500 μg per plate. In the confirmatory assay (Experiment 23500-C1, Tables 5 and 6), all data were acceptable, and no positive
increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.
All criteria for a valid study were met.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Rangefinding Study
Experiment ID 23500 -A1	Date Plated: 01 -Feb-02
Vehicle: DMSO	Date Counted:12 -Feb-02
	TA100 Revertants Per Plate
	With S9		Without S9
ug/Plate	RevertrantsPer Plate	Background LawnEvaluation^a	RevertantsPerPlate	BackgroundLawnEvaluation^a
0.00 (Vehicle , 50uL)	158	N	126	N
Test Article 6.67	115	N	125	N
10.0	134	N	134	N
33.3	116	N	132	N
66.7	93	N	133	N
100	111	N	136	N
333	96	R	0	R
667	62	R	0	A
1000	0	A	0	A
3330	0	A	0	A
5000	0	A	0	A
^aBackground Lawn Evaluation Codes: N = normal, R = reduced, A = Absent, P = precipitate, o = obscured by percipitate

Table 2: Rangefinding Study
Experiment ID 23500 -A1	Date Plated: 07 -Feb-02
Vehicle: DMSO	Date Counted: 12 -Feb-02
	WP2uvrA Revertants Per Plate
	With S9		Without S9
ug/Plate	RevertrantsPer Plate	Background LawnEvaluation^a	RevertantsPerPlate	BackgroundLawnEvaluation^a
0.00 (Vehicle , 50uL)	25	N	23	N
Test Article 6.67	21	N	14	N
10.0	10	N	12	N
33.3	18	N	18	N
66.7	27	N	14	N
100	20	N	17	N
333	16	R	0	R
667	0	R	0	R
1000	0	R	0	R
3330	0	A	0	R
5000	0	A	0	A
^aBackground Lawn Evaluation Codes: N = normal, R = reduced, A = Absent, P = precipitate, o = obscured by percipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative Negative - with and without S-9 metabolic activation

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay
indicate that under the conditions of this study, the test article, Diisobutyl Carbinol, did not cause a positive increase in the mean number of
revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor -induced rat
liver (S9).