Alcohols, C13-15-branched and linear

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2021 - July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Aug 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 Jun 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C13-15-branched and linear

EC Number:

287-625-4

EC Name:

Alcohols, C13-15-branched and linear

Cas Number:

85566-16-1

Molecular formula:

C13 H27 OH - C15 H31 OH

IUPAC Name:

Alcohols C13-C15 branched and linear, reaction products of C12-C14 olefines

Test material form:

liquid

Details on test material:

Name of test substance: C13-C15-Alcohol
Test substance No.: 20/0140-2
Batch identification: TK2004-02122020
CAS No.: 85566-16-1
Identity: confirmed
Purity: 100% UVCB
Water content: 0.01 g/100 g
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
Storage stability: The stability of the test substance under storage conditions is guaranteed until 02 Dec 2021 as indicated by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
Date of production: 02 Dec 2020
Physical state, appearance: liquid, colorless, clear
Storage conditions: room temperature

Specific details on test material used for the study:

The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
The test substance was dissolved in dimethyl sulfoxide (DMSO).
To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
The further concentrations were diluted according to the planned doses.
All test substance formulations were prepared immediately before use.

The stability of the test substance in the vehicle DMSO was not determined analytically, because the test substance was administered immediately after preparation and is usually stable.

Method

Species / strain

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction
The S9 fraction was prepared according to Ames et al. at BASF SE in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and beta-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am.
Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

S9 mix
The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.
To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.

Test concentrations with justification for top dose:

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

Vehicle / solvent:

Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

yes

Remarks:

Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strain.

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains. Vehicle controls were used for several BASF projects done in parallel.

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other:

Remarks:

The stability of the selected positive controls was well-defined under the selected culture conditions, since they were well-established reference mutagens. Positive controls were used for several BASF projects done in parallel.

Details on test system and experimental conditions:

Test strain
For testing, a deep-frozen (-70°C to -80°C) bacterial culture (E. coli WP2 uvrA) is thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria culture was determined-Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). This culture grown overnight was kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strain mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
E. coli WP2 uvrA was checked for UV sensitivity.
Tryptophan auxotrophy was checked in each experiment via the spontaneous rate.

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al..
• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager.
Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al..
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Rationale for test conditions:

Scope of tests and test conditions
1st Experiment
Strain: E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control
2nd Experiment
Strain: E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

Evaluation criteria:

Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

Toxicity
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Solubility
If precipitation of the test material was observed, it would be recorded and indicated in the tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Results and discussion

Test results

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY AND STERILITY CONTROL
Test substance precipitation was observed at and above 2500 μg/plate with and without S9 mix.
The additional treated plates for sterility control showed no contamination in all performed experiments.

Any other information on results incl. tables

For detailed result tables and historical control data see "Attached background material"

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that C13-C15-Alcohol is not a
mutagenic test substance in the bacterial reverse mutation test in the absence and the
presence of metabolic activation.