Reaction products of long-chain fatty alcohols and inorganic acid with 2-aminoalkanol

Genetic toxicity: in vitro

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Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From 2013, October 14 to 2013, November 21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

yes

Remarks:

The study integrity is not affected by the deviations

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Method

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:

S9-mix

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate were tested in triplicate. The highest concentration of the test material used in the subsequent mutation assay was 5000 μg/plate.

Vehicle / solvent:

A solubility test was performed in a previous project. The test substance could not be dissolved in water or in dimethyl sulfoxide since no workable suspensions were obtained. Since the test substance formed in ethanol a workable suspension, ethanol was selected as solvent. At concentrations of 10 mg/ml and higher test substance formed a hazy suspension in ethanol (Merck, Darmstadt, Germany). At concentrations of 3.33 mg/ml and lower the test substance was dissolved in ethanol.

Controls

Untreated negative controls:

yes

Remarks:

The vehicle of the test substance, which was ethanol

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2AA (2-aminoanthracene for all strains with metabolic activation) ICR-191 for TA1537 without metabolic activation

Details on test system and experimental conditions:

Cell culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Merck) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Merck) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Acros Organics).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Merck) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3 °C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0 °C (actual range 36.4 – 37.7°C) in the dark.

Metabolic activation system
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg), except in the second experiment in tester strain WP2uvrA. In this part of the experiment, rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany (deviation 1).

Preparation of S9-fraction
The animals were housed at WIL Research Europe in a special room under standard laboratory conditions, as described in the Standard Operating Procedures. The rats were orally dosed for three consecutive days with a suspension of phenobarbital (Bufa B.V., IJsselstein, The Netherlands) (80 mg/kg body weight) and ß-naphthoflavone (Sigma Aldrich Chemie) (100 mg/kg body weight) in corn oil (Fagron Farmaceutical, Capelle aan de IJssel, The Netherlands) (they were denied access to food for 3 to 4 hours preceding each dosing). One day after the final exposure (24 h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA (Merck). Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampoules, which were stored in liquid nitrogen (-196 °C) for a maximum of 1 year. Before use, all S9 batches were characterised with the mutagens Benzo-(a)-pyrene (Sigma) and 2-aminoanthracene (Sigma), which require metabolic activation, in tester strain TA98 at concentrations of 5 µg/plate and 1 µg/plate, respectively.

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9- fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Study design
Dose range finding test: selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. The highest concentration test material used in the subsequent mutation assay was 5000 µg/plate.

Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. In the first experiment test substance was tested both in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted.

Colony counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test article precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme:
- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA).
-  Ames study Manager version 1.23 (Perceptive Instruments Ltd.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC).

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Statistics:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

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

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

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:

no cytotoxicity

Vehicle controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in the tester strains TA1535 and TA1537 in the absence of S9-mix at the highest tested concentration and a biologically relevant decrease in the number of revertant colonies in tester strain TA1537 in the presence of S9-mix at the highest tested concentration.
In an independent repeat of the assay with additional parameters, the test substance was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. No or a slight precipitate of test material was observed at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all tester strains, except in tester strain TA1535 in the presence of S9-mix and WP2uvrA in the absence and presence of S9-mix.

Any other information on results incl. tables

Deviations from the guideline

1. In the mutation experiment (except in experiment 2, WP2uvrA), the S9-fraction was obtained from Trinova Biochem.

Evaluation: Due to a shortage of the S9 homogenate prepared at WIL Research Europe, S9 homogenate obtained from Trinova Biochem was used. The use of another S9 homogenate had no effect on the results of the study.

2. In the first experiment, the mean plate counts of the solvent control of TA1535 in the absence and presence of S9-mix were not within the laboratory historical range.

Evaluation: Mean plate counts of 26 and 31 revertants (in the absence and presence of S9-mix, respectively) were just above the limit of the range (25), therefore this deviation in the mean plate count of the solvent controls had no effect on the results of the study.

3. Fifteen μg/plate 2-amino anthracene was used as positive control substance in the presence of S9-mix for tester strain WP2uvrA.

Evaluation: To be sure that a clear positive response was obtained, the concentration of the positive control substance was increased. This had no influence on the study integrity.

4. The positive control substances of the following tester strains: tester strain TA1535 (absence of S9-mix, first and second experiment), TA100 (presence of S9-mix, second experiment) and WP2uvrA (absence of S9-mix, dose range finding test and second experiment) showed responses (mean plate count) which were not within the laboratory historical range.

Evaluation: The values were above the limit of the range. These values were more than 3 times greater than the concurrent solvent control values, therefore this deviation in the mean plate count of the positive controls had no effect on the results of the study.

5. In the dose range finding test, the mean plate count of the positive control of WP2uvrA in the presence of S9-mix was not within the laboratory historical range.

Evaluation: Although the mean value of 99 was below the limit of the range (122), the mean value was more than 3 times greater than the concurrent solvent control value, therefore this deviation in the mean plate count of the positive control had no effect on the results of the study.

6. The experimental end date was 21 November 2013, which is not within the period mentioned in the time schedule in the protocol.

Evaluation: This deviation in the experimental end date had no effect on the results of the study.

Applicant's summary and conclusion

Conclusions:

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) or in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Executive summary:

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

At concentrations of 10 mg/ml and higher the test substance was suspended in ethanol. At concentrations of 3.33 mg/ml and lower the test substance was dissolved in ethanol.

In the dose range finding test, the substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 3330 and 5000 μg/plate. Toxicity was only observed in tester strain TA100 in the absence and presence of S9-mix at dose levels of 3330 and 5000 μg/plate.

Based on the results of the dose range finding test,the substance was tested in the first mutation assay at a concentration range of 100 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in the tester strains TA1535 and TA1537 in the absence of S9-mix at the highest tested concentration and a biologically relevant decrease in the number of revertant colonies in tester strain TA1537 in the presence of S9-mix at the highest tested concentration.

In an independent repeat of the assay with additional parameters, the substance was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. No or a slight precipitate of the test material was observed at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all tester strains, except in tester strain TA1535 in the presence of S9-mix and WP2uvrA in the absence and presence of S9-mix.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) or in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

The negative control values were within the laboratory historical control data ranges, except for TA1535 in the first experiment. However, since these values were just outside the limit of the range, the validity of the test was considered to be not affected. The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate.

Based on the results of this study it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.