Phosphoric acid, C11-14 (C13-rich)-isoalkyl mono- and di-esters, partially salted with 1-Propanamine, 3-(tridecyloxy)-, branched

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:

29 June 2016 to 18 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

study report issued later than planned with no impact on results or integrity of the investigation

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

study report issued later than planned with no impact on results or integrity of the investigation

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

study report issued later than planned with no impact on results or integrity of the investigation

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

yes

Remarks:

study report issued later than planned with no impact on results or integrity of the investigation

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Confidential

IUPAC Name:

Confidential

Test material form:

liquid

Details on test material:

- Appearance/physical state: Amber coloured liquid
- Storage conditions: Room temperature in the dark

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from the livers of phenobarbital / β-naphthoflavone induced rats

Test concentrations with justification for top dose:

RANGE-FINDING TEST
- Test item concentrations: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate.

INITIAL MUTATION TEST AND CONFIRMATORY MUTATION TEST (Salmonella typhimurium TA98, TA100, TA1535 and TA1537)
- Test item concentrations: 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.

INITIAL MUTATION TEST (Escherichia coli WP2 uvrA )
- Test item concentrations: 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.

CONFIRMATORY MUTATION TEST (Escherichia coli WP2 uvrA )
- Test item concentrations: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate,

COMPLEMENTARY CONFIRMATORY MUTATION TEST (Salmonella typhimurium TA1535 with metaabolic activation)
- Test item concentration: 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.

Vehicle / solvent:

N,N-dimethylformamide (DMF)

Controls

Controls 1

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylenediamine

Remarks:

4 µg/plate for Salmonella TA98 without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2 µg/plate for Salmonella TA100 and TA1535 without metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate for Salmonella TA1537 without metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

other: methyl methanesulphonate

Remarks:

2 µL/plate for E coli WP2 uvrA without metabolic activation

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2 µg/plate for all Salmonella strains with metabolic activation

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

50 µg/plate for E coli WP2 uvrA with metabolic activation

Controls 7

Negative solvent / vehicle controls:

yes

Remarks:

N,N-dimethylformamide

Remarks:

Sigma Aldrich; batch SZBE1830V (purity 100 %; expiry date 16 June 2017) and batch 15K060521 (purity 99.9 %;expiry date 31 October 2020)

Controls 8

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Remarks:

TEVA Hungary Co (batch 7170914; expiry date 30 September 2017)

Controls 9

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (water ≤ 0.02 %)

Remarks:

Sigma Aldrich batch SZBF3070V (purity 99.99 %; expiry date 18 October 2018) and batch SZBG0550V (purity 99.98 %; expiry date 08 February 2019)

Details on test system and experimental conditions:

FORMULATION
- The appropriate vehicle and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test.
- The vehicle N,N-dimethylformamide (DMF) was used to prepare the stock formulation of the test material.
- Test formulations were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock formulation using the selected vehicle.
- The respective test concentrations in the main tests are shown in Table 1 (below).
- Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

POSITIVE CONTROLS
- Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.
- Positive control materials were selected based on the scientific literature, the experience of the Test Facility and the availability of historical control data.

VEHICLE/SOLVENT CONTROLS
- Three vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals.
- DMSO (batch SZBF3070V) was used in the preliminary concentration range finding test.
- DMF (batch 15K060521) was used in the complementary confirmatory mutation test.

BACTERIAL STRAINS
- Source: All bacterial strains were received from MOLTOX (Molecular Toxicology Inc, Boone, North Carolina, USA) on 21 April 2015. True copies of original certificates plus other strain documentation were collected and stored in the Microbiological Laboratory of CiToxLAB Hungary Ltd.
- Genotypes: In addition to histidine or tryptophan mutation, each strain has additional mutations,
which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules.The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair. The genotypes of the tester strains used for mutagenicity testing are summarized in Table 3 (attached).
- Storage: The strains were stored at -80 ± 10ºC in the Culture Collection of the Microbiological
Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- Confirmation of phenotype: The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly
according to Ames et al. and Maron and Ames. Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of CiToxLAB Hungary Ltd.

SPONTANEOUS REVERSION OF TESTER STRAINS
- Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (in Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
- Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011 to 2014 were (as guide) as follows: Salmonella typhimurium TA98: 9-46, TA100: 54-210, TA1535: 1-46, TA1537: 1-24, Escherichia coli WP2 uvrA: 11-82.
PROCEDURE FOR GROWING CULTURES
- The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum was used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay.
- The cultures were incubated for 10-14 hours at 37 °C in a gyrotory water bath shaker.

VIABILITY OF THE TESTING CULTURES
- The viability of each testing culture was determined by plating 0.1 mL of the 10E05, 10E06, 10E07 and 10E08 dilutions prepared by sterile physiological saline on nutrient agar plates.
- The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

MEDIA
- The supplier, batch number and expiry date of chemicals used in the investigation is summarised in Table 6 (attached).
- Minimal glucose agar typically contained glucose (20.0 g/L); magnesium sulfate (0.2 g/L); citric acid (2.0 g/L); dipotassium hydrogenphosphate (10.0 g/L); sodium ammonium hydrogenphosphate (3.5 g/L); agar (13.0 g/L); distilled water of a quantity sufficient to give a volume of 1000 mL.
- Merck minimal glucose agar plates (batch 138348; expiry date 06 September 2016) was used in the preliminary, batch 138706 (expiry date 27 September 2017) was used in the confirmatory mutation test and batch number 139474 (expiry 13 November 2016) was used in the complementary confirmatory mutation test. Certificates of Analysis were obtained from the supplier.
- Nutrient Broth No 2 (25.0 g made up to a volume of 1000 mL with distilled water) was sterilised in an autoclave at 121 °C.
- Nutrient agar (20.0 g made up to a volume of 1000 mL with distilled water) was sterilised in an autoclave at 121 °C.
- Agar solution contained agar bacteriological (4.0 g); NaCl (5.0 g); sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed at 121 °C in an autoclave.
- Histidine-biotin solution (0.5 mM) contained D-biotin (FW 244.31) 122.2 mg; L-histidine HCl:H2O (FW 209.63) 104.8 mg; sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed by filtration using a 0.22 µm membrane filter.
- Complete top agar for Salmonella typhimurium strains contained 0.5 mM histidine-biotin solution (100 mL) and agar solution (900 mL).
- Trptophan solution (2 mg/mL) contained L-tryptophan (FW 204.23) 2000 mg and sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed by filtration using a 0.22 µm membrane filter.
- Complete top agar for the Escherichia coli strain contained Nutrient Broth No 2 (50 mL), 2 mg/mL tryptophan solution (2.5 mL) and agar solution 947.5 mL.

METABOLIC ACTIVATION SYSTEM
- Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
- The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of CiToxLAB Hungary Ltd. according to Ames et al. and Maron and Ames.
- The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
- The supplier, batch number and expiry date of chemicals used are summarized in Table 6 (attached).

INDUCTION OF LIVER ENZYMES
- Male Wistar rats (385-450 g, animals were 11 weeks old at the initiation) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days.
- Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed.
- Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels.
- Initiation of the induction of liver enzymes used for preparation S9 used in this study was 25 January 2016.

PREPARATION OF RAT LIVER HOMOGENATE S9 FRACTION
- On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools.
- After excision, livers were weighed and washed several times in 0.15 M KCl.
- The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.
- Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained.
- The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10 ºC.
- The date of preparation of S9 fraction for this study was 28 January 2016 (CiToxLAB code: E12297).
- The sterility of the preparation was confirmed. The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd.
- The mean protein concentration of the S9 fraction used was determined to be 30.8 g/L.
- The biological activity in the Salmonella assay of S9 was characterized using the two mutagens (2-Aminoanthracene and Benzo(a)pyrene) that requires metabolic activation by microsomal enzymes.
- The batch of S9 used in this study functioned appropriately.

S9 MIX
- Salt solution for the S9 mix contained NADP Na (7.66 g); D-glucose-6-phosphate Na (3.53 g); MgCl2.6H20 (4.07 g); KCl (6.15 g); sufficient distilled water to give 1000 mL. Sterilisation was performed by filtration through a 0.22 µm membrane filter.
- Sodium phosphate buffer 0.2 M (pH 7.4) solution A contained Na2HPO4 (71.63 g) and sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed in an autoclave at 121 °C.
- Sodium phosphate buffer 0.2 M (pH 7.4) solution B contained Na2PO4.H2O (27.6 g) and sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed in an autoclave at 121 °C.
- Sodium phosphate buffer pH 7.4 contained solution A (880 mL) and solution B (120 mL).
- The complete S9 mix was freshly prepared and contained ice cold 0.2 M sodium phosphate buffer pH 7.4 (500 mL); S9 rat liver homogenate (100 mL); salt solution for S9 mix (400 mL).
- The S9 mix was kept in an ice bath prior to addition to the culture medium.

TEST PROCEDURE
- The study included a preliminary compatibility test, a preliminary range fnding test (informatory toxicity test), an initial mutation test, a confirmatory mutation test and a complementary confirmatory mutation test.
- At the request of the Sponsor the plate incorporation method was used during the whole study.

CONCENTRATIONS
- Concentrations were selected on the basis of the preliminary compatibility test and preliminary range finding test (informatory toxicity test).

PRELIMINARY COMPATIBILITY TEST
- Solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF).
- The test item was insoluble at 100 mg/mL concentration using Distilled water. Partial dissolution was observed at the same concentration using DMSO as vehicle. However the formulation at the same concentration using DMF was a solution and was suitable for the test. Therefore DMF was selected as vehicle for the test.
- The obtained stock formulation (50 μL) with the solution of top agar (5.4.4.) and phosphate buffer was examined in a test tube without test bacterium suspension. The results of the Solubility Test are summarized in Table 4 (below).

PRELIMINARY CONCENTRATION RANGE-FINDING TEST (INFORMATORY TOXICITY TEST)
- Based on the solubility test, 100 mg/mL stock formulation was prepared in DMF which was diluted in 6 steps by factors of 2, 2.5 and approximately √10.
- The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the
tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item.
- In the preliminary concentration range finding test the plate incorporation method was used.

TEST ITEM CONCENTRATIONS IN THE MUTAGENICITY TESTS
- Based on the results of the preliminary tests, 100 mg/mL stock formulation was prepared from the test item with DMF, which was diluted by serial dilutions in several steps to obtain nine or ten dosing formulations.
- The maximum test concentration was 5000 or 1581 or 500 μg test item/plate in the initial mutation test and confirmatory mutation tests.
- Examined concentrations in the initial mutation test and confirmatory mutation test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate.
- Examined concentrations in the initial mutation test in the Escherichia coli WP2 uvrA strain were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate.
- Examined concentrations in the confirmatory mutation test in the Escherichia coli WP2 uvrA strain were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain.
- Examined concentrations in the complementary confirmatory mutation test were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581μg test item/plate for the Salmonella typhimurium TA1535 bacterial strain with metabolic activation.

CONTROL GROUPS USED IN THE TESTS
- Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test.
- In addition, untreated control was used demonstrating that the chosen vehicle (solvent) induced no deleterious or mutagenic effects.
- The control groups are summarised in Table 5 (below).
- If the solvent of the positive control substance differed from the vehicle (solvent) of the test item, both solvents were run in the assay.

EXPOSURE PROCEDURE
- A standard plate incorporation procedure was performed, as an initial mutation test, confirmatory mutation test and complementary confirmatory mutation test.
- Bacteria (cultured in Nutrient Broth No. 2 were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
- Molten top agar was prepared and kept at 45 °C.
- Top agar (2 mL) was aliquoted into individual test tubes (3 tubes per control or concentration level).
- The equivalent number of minimal glucose agar plates were properly labelled.
- The test item and other components were prepared freshly and added to the overlay (45 °C).
- Tubes contained top agar (2000 µL); vehicle (solvent) or test formulation or reference controls (50 µL); overnight culture of test strain (100 µL); phosphate buffer (pH 7.4) or S9 mix (500 µL).
- The solution was mixed and poured on the surface of minimal agar plates.
- For studies with metabolic activation, instead of phosphate buffer, the S9 mix (0.5 mL) was added to each overlay tube.
- The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls.
- After preparation, the plates were incubated at 37 °C for 48 hours.

EVALUATION OF EXPERIMENTAL DATA
- Colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting.
- Visual examination of the plates was also performed and precipitation or signs of growth inhibition (if any) were recorded and reported.
- The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft Excel software.
- Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Evaluation criteria:

VALIDITY CRITERIA
- The number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests.
- At least five analysable concentrations were presented in all strains of the main tests.

CRITERIA FOR A POSITIVE RESPONSE
- A dose–related increase in the number of revertants occurred and/or a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
- An increase was considered biologically relevant if:
(i) The number of reversions was more than two times higher than the reversion rate of the
negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains.
(ii) The number of reversions was more than three times higher than the reversion rate of the
negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial
strains.
- According to the guidelines, statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

CRITERIA FOR A NEGATIVE RESPONSE
- A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

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 2

Key result

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 3

Key result

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 4

Key result

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 5

Key result

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:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
- In the preliminary range finding test, the plate incorporation method was used.
- The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (vehicle/solvent) and positive controls.
- In the test each sample (including the controls) was tested in triplicate.
- In the range finding test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
- Inhibitory, cytotoxic effects of the test item (absent/reduced and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the preliminary range finding test in Salmonella typhimurium TA98 and TA100 strains without metabolic activation at 5000, 2500, 1000, 316 and 100 μg/plate concentrations and in both strains with metabolic activation at 5000, 2500, 1000 and 316 μg/plate concentrations.
- No precipitate of the test item was detected in the Preliminary Range Finding Test.
- Plates had whitish discoloration at the concentration of 5000 μg/plate in both tested strains with and without metabolic activation.
- The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 and Appendix 3 (attached).

INITIAL AND CONFIRMATORY MUTATION TESTS
- In the Initial mutation test on Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation no inhibitory or cytotoxic effect of the test item was observed.
- According to the test guidelines the maximum test concentration for soluble non-cytotoxic substances should be 5000 µg test item/plate. Therefore the initial mutation test on Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation were considered to be invalid and was repeated to examine the 5000 µg test item/plate concentration as well. Results of the invalid experiment on this strain are not reported; however, all data were kept and archived in the raw data binder.
- The repeated test with Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation was considered as part of the initial mutation test.
- The confirmatory mutation test on Salmonella typhimurium TA1535 bacterial strain with metabolic activation did not meet the validity criteria; therefore, an additional experiment (complementary confirmatory mutation test) was performed in this strain in an additional experimental period (Experimental Period III) to complete the data. The experimental conditions (except concentrations) were the same as in the confirmatory mutation test. Results of the invalid experiment were not reported; however, all data was kept and archived in the raw data binder.
- In the initial mutation test and confirmatory mutation test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.
- In the initial mutation test, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain without metabolic activation at the concentration of 5 µg/plate. The mutation factor value was 1.62. However, there was no dose response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- In the confirmatory mutation tests, the highest revertant rate was observed in Escherichia coli WP2 uvrA bacterial strain without metabolic activation at 1.581 µg/plate concentration. The mutation factor value was 1.31. However, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- Slightly higher numbers of revertant colonies compared to the solvent control were detected in the initial mutation test and in the confirmatory mutation tests sporadically. However, the numbers of revertant colonies were below the biologically relevant threshold value in all cases, so they were considered as reflecting the biological variability of the test.
- Slightly lower revertant counts compared to the solvent control were observed in the initial mutation test and in the confirmatory mutation tests at some non-cytotoxic concentrations. However, the mean numbers of revertant colonies were in the historical control range in all cases.
- Inhibitory, cytotoxic effects of the test item (absent/reduced/slightly reduced background lawn development and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the initial mutation test in Salmonella typhimurium TA98 and TA1535 strains at 500 and 158.1 μg/plate concentrations with and without metabolic activation, in Salmonella typhimurium TA100 and TA1537 strains at 500, 158.1 and 50 μg/plate concentrations without metabolic activation, in these strains at 500 and 158.1 μg/plate concentrations with metabolic activation.
- Similarly, inhibitory, cytotoxic effects of the test item (absent/reduced/slightly reduced background lawn development and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the confirmatory mutation test and complementary confirmatory mutation test in Salmonella typhimurium TA98 strain at 500 and 158.1 μg/plate concentrations without metabolic activation, in this strain at 500 μg/plate concentration with metabolic activation, in Salmonella typhimurium TA100 and TA1537 strains at 500, 158.1 and 50 μg/plate concentrations without metabolic activation, in these strains at 500 and 158.1 μg/plate concentrations with metabolic activation, in Salmonella typhimurium TA1535 strain at 5000, 1581, 500 and 158.1 μg/plate concentrations with and without metabolic activation.
- No precipitate was detected on the plates in the main tests in any examined bacterial strain with and without metabolic activation.
- The experimental results (revertant colony numbers per plate, mutation factors, and standard deviations) are summarized in Tables 8-9 and Appendices 4, 5 and 6 (attached).

VALIDITY OF THE TESTS
- Untreated, negative (vehicle/solvent) and positive controls were run concurrently.
- The mean values of revertant colony numbers of untreated, negative (vehicle/solvent) and positive control plates were within the historical control range.
- At least five analysable concentrations were presented in all strains of the main tests.
- The reference mutagens showed a distinct increase of induced revertant colonies.
- The viability of the bacterial cells was checked by a plating experiment in each test.
- The tests were considered to be valid.

Remarks on result:

other: No precipitate was detected on the plates

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions applied the test item did not induce gene mutations by basepair changes or frameshifts in the genome of the strains used. The test item demonstrated no mutagenic activity in the applied bacterial tester strains under the test conditions used in the study.

Executive summary:

GUIDELINE

The test item was investigated for potential mutagenic activity using the bacterial reverse mutation assay. The study was conducted in accordance with the Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No 471, "Bacterial Reverse Mutation Test" (21 July 1997), EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", EPA 712-C-98-247 (August 1998) and Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria" (30 May 2008). The method also conformed to conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

 

METHODS

Experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.

 

The study included a preliminary solubility test, a preliminary range-finding test (informatory toxicity test), an initial mutation test, a confirmatory mutation test and a complementary confirmatory mutation test. The plate incorporation method was used throughout the study.

 

Based on the results of the solubility test and available information, the test item was formulated in N,N-dimethylformamide (DMF). Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the range finding test. Examined concentrations in the initial mutation test and confirmatory mutation test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains were 500, 158.1,50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate. Examined concentrations in Escherichia coli WP2 uvrA strain in the initial mutation test were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate and in the confirmatory mutation test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate. Examined concentrations in the complementary confirmatory mutation test were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/platein Salmonella typhimurium TA1535 bacterial strain with metabolic activation.

 

RESULTS

In the initial mutation test and confirmatory mutation tests, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends.

 

Inhibitory, cytotoxic effects of the test item were observed in the initial mutation test and repeatedly in the confirmatory mutation test and complementary confirmatory mutation test for Salmonella typhimurium bacterial strains in the higher concentration range.

 

The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

 

CONCLUSION

Under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item demonstrated no mutagenic activity in the applied bacterial  tester strains under the test conditions used in the study.