2-propylheptan-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Test material information

Constituent 1

Reference substance name:

2-propylheptan-1-ol

EC Number:

233-126-1

EC Name:

2-propylheptan-1-ol

Cas Number:

10042-59-8

Molecular formula:

C10H22O

IUPAC Name:

2-propylheptan-1-ol

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Propylheptan-1-ol
- Physical state: Clear liquid
- Analytical purity: 96.20 area %
- Isomers composition: isomers of C10 alcohols 2.94 area %
- Lot/batch No.: 16.06.2008
- Test substance No.: 0649/82526
- Expiration date of the lot/batch: July 2009
- Storage condition of test material: Room temperature

Method

Target gene:

his- and trp-gene

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

5 - 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: 2-propylheptan-1-ol was insoluble in water. Its solubility was assessed at 50 mg/mL in dimethyl sulphoxide (DMSO) in which it was dissolved. DMSO was, therefore, used as the vehicle for this study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see: Details on test system and conditions

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

First test:

Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or negative control were placed in glass vessels. The negative control was the chosen vehicle, DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter. Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four nonprecipitating concentrations should be obtained, unless otherwise justified by the Study Director.

Second test:

As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, and seven concentrations were
used in order to ensure that at least four non-toxic concentrations were tested.

Positive controls:

In the absence of S9 mix:
- Sodium azide (in DMSO): 2 μg/plate for strains TA100 and TA1535
- 9-Aminoacridine (in DMSO): 50 μg/plate for strain TA1537
- 2-Nitrofluorene ( in DMSO): 2 μg/plate for strain TA98
- 4-Nitroquinoline-1-oxide (in DMSO): 2 μg/plate for strain WP2 uvrA (pKM101)

In the presence of S9 mix:
- 2-Aminoanthracene (in DMSO): 5 μg/plate for strains TA100 and TA1535, 10 μg/plate for strain WP2 uvrA (pKM101)
- Benzo[a]pyrene: (in DMSO): 5 μg/plate for strains TA98 and TA1537

Evaluation criteria:

Acceptance criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.

Criteria for assessing mutagenic potential
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important.

Statistics:

The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium: strains TA1535, TA1537, TA98 and TA100; Escherichia coli: strain WP2 uvrA (pKM101)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(in all strains at one or more concentrations in both tests)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Any other information on results incl. tables

1st experiment:Standard plate test (5 - 5000 µg/plate)
Strain	Metabolic activation system	mean his^+revertant colonies (negative control)	maximum revertant factor (conc. (µg/plate))	dose dependency	Assessment	maximum revertant factor (positive control)	comments
TA 98	no	36.7	1.1 (50 / 500)	no	negative	9 (2NF)	Thinning of background lawn at 5000 µg/plate
	yes	9.3	1.0 (5 / 500 / 1500)	no	negative	3.3 (B[a]P)	Slight thinning of background lawn at 5000 µg/plate
TA 100	no	150	1.0 (15 / 50)	no	negative	4.3 (NaN3)	Slight thinning of background lawn at 5000 µg/plate
	yes	178	1.0 (50)	no	negative	15.7 (AAN)	Slight thinning of background lawn at 5000 µg/plate
TA 1537	no	8.7	1.4 (150)	no	negative	40.3 (AAC)	Slight thinning of background lawn at 1500 µg/plate; Thinning of background lawn at 5000 µg/plate;
	yes	32	0.9 (15 / 50 / 150 / 500)	no	negative	4.6 (B[a]P)	Slight thinning of background lawn at 1500 µg/plate; Severe thinning of background lawn at 5000 µg/plate;
TA 1535	no	26.7	1.2 (50)	no	negative	43.6 (NaN3)	Slight thinning of background lawn at 1500 µg/plate; Thinning of background lawn at 5000 µg/plate;
	yes	28	0.9 (15)	no	negative	14.8 (AAN)	Thinning of background lawn at 5000 µg/plate
WP2 uvrA	no	117.3	1.3 (500)	no	negative	26.6 (NQO)	Thinning of background lawn at 5000 µg/plate;
	yes	171	1.2 (500)	no	negative	2.5 (AAN)	Slight thinning of background lawn at 5000 µg/plate
2nd experiment:Preincubation test (5 - 5000 µg/plate)
Strain	Metabolic activation system	mean his^+revertant colonies (negative control)	maximum revertant factor (conc. (µg/plate))	dose dependency	Assessment	maximum revertant factor (positive control)	comments
TA 98	no	36.7	1.0 (5 / 15 / 50 / 150)	no	negative	7.5 (2NF)	Slight thinning of background lawn at 500 µg/plate; Severe thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
	yes	48.7	1.1 (50)	no	negative	8.2 (B[a]P)	Slight thinning of background lawn at 500 µg/plate; Severe thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
TA 100	no	174.7	1.1 (5)	no	negative	5.1 (NaN3)	Slight thinning of background lawn at 500 µg/plate; Severe thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
	yes	197	0.9 (5 / 15)	no	negative	14.6 (AAN)	Slight thinning of background lawn at 500 µg/plate; Severe thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
TA 1537	no	12	0.9 (5)	no	negative	87.2 (AAC)	Thinning of background lawn at 500 µg/plate; Lawn absent at 1500 and 5000 µg/plate;
	yes	30.7	1.0 (5)	no	negative	7.6 B[a]P)	Thinning of background lawn at 500 µg/plate; Lawn absent at 1500 and 5000 µg/plate;
TA 1535	no	22	1.2 (15)	no	negative	54.5 (NaN3)	Thinning of background lawn at 500 µg/plate; Lawn absent at 1500 and 5000 µg/plate;
	yes	26	1.1 (15)	no	negative	13.1 (AAN)	Thinning of background lawn at 500 µg/plate; Lawn absent at 1500 and 5000 µg/plate;
WP2 uvrA	no	178.3	1.0 (5)	no	negative	18 (NQO)	Thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;
	yes	206.3	1.0 (5 / 50)	no	negative	2.9 (AAN)	Thinning of background lawn at 1500 µg/plate; Lawn absent at 5000 µg/plate;

Applicant's summary and conclusion

Conclusions:

According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce back mutations in selected loci in several strains of Salmonella typhimurium and one Escherichia coli strain in the Ames test.

 

Strains:                       TA 1535, TA 100, TA 1537, TA 98, Escherichia coli WP2 uvrA

Dose range:                15 µg - 5,000 µg/plate (SPT); 15 µg -  250 µg/plate (PIT)

Test conditions:           Standard plate test (SPT) and pre­ incubation test (PIT) both with and without metabolic activation (Aroclor induced rat liver S-9 mix).

Solubility:                    No precipitation of the test sub­stance was found.

Toxicity:                     A bacteriotoxic effect was observed depending on the strain and test conditions from about 120 µg - 250 µg/plate onward.

 

 

Mutagenicity:

An  increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mutagenicity:

An  increase in the number of his• revertants was not observed both in the standard plate test

andin the preincubation test either without S-9 mix or after the addition of a metabolizing system.