Octadecene, reaction products with hexadecene, hydrogenated

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:

09 April 2019 to 09 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, adopted 21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, B.13/14. “Mutagenicity: Reverse Mutation Test Using Bacteria”, 30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse Mutation Test”, EPA 712-C-98-247, August 1998

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:

Oligomerisation products of alpha-alkenes C16-18 (even numbered), hydrogenated, hydroisomerised

EC Number:

832-827-5

Cas Number:

2241366-04-9

Molecular formula:

Variable - UVCB

IUPAC Name:

Oligomerisation products of alpha-alkenes C16-18 (even numbered), hydrogenated, hydroisomerised

Test material form:

liquid

Details on test material:

Name: SynNova Base Oil
CAS number: 2241366-04-9
Batch/Lot number: TS20371/TS21270
Description: Liquid - water white colorless oil.
Purity: 100%
Expiry date: 25 February 2021/31 July 2021
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

histidine (his) and tryptophan (trp)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

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 composition of solution refers to 1000 mL.

Induction of Liver Enzymes
Male Wistar rats (349-395 g, animals were 9-10 weeks old at 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 27 November 2018 (Citoxlab code: E13013).

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 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 30 November 2018 (Citoxlab code: E13013, Expiry date: 30 November 2020).
The sterility of the preparation was confirmed. The protein concentration of the preparation was determined by a chemical analyzer 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 26.7 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.

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Range Finding Test. In Assay 1 and Assay 2 the same concentrations were used based on the absence of positive effect of the test item.
Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1, 2 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.

Vehicle / solvent:

Propylene Glycol (PG) + 2% Polysorbate 80 was used as solvent to prepare the stock solution of the test material. The test item was freshly formulated at a concentration of 100 mg/mL in the vehicle on the day of administration in the Pharmacy of Citoxlab Hungary Ltd. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent. The formulation container was magnetic stirred continuously up to the end of dose administration procedures.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO), Distilled water & Propylene glycol with 2% (w/v) Polysorbate 80

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2AA) & 4-nitro-1,2-phenylene-diamine (NPD)

Details on test system and experimental conditions:

DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, an Assay 1 and an Assay 2. In the Preliminary Range Finding Test as well as in the Assay 1, the plate incorporation method was used. In Assay 2 the plate incorporation method or the pre-incubation method was used.

Preliminary Compatibility Test
Based on the available information (trial formulations of the test item performed at the Test Facility), the test item was insoluble in the generally used vehicles (distilled water, DMSO, Acetone, Ethanol, PG + 1% Polysorbate 80). At the concentration level of 200 mg/mL settling emulsion was detected in Propylene Glycol + 2% Polysorbate 80. This emulsion with continuously stirring was suitable for the test. Therefore, PG + 2% Polysorbate 80 was selected for vehicle (solvent) of the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, a 100 mg/mL stock solution was prepared in PG + 2% Polysorbate 80. Six test concentrations were prepared by successive dilutions of the stock solution, spaced 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 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Assay 1 and Assay 2)
Based on the results of the preliminary test and main tests, in the Assay 1, 2 a 100 mg/mL stock solution was prepared in PG + 2% Polysorbate 80, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.

Control Groups Used in the Tests
Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Procedure for Exposure in the Assay 1
Assay 1 followed the standard plate incorporation procedure. 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. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle or test item formulation (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix 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±1 hours.

Procedure for Exposure in the Assay 2
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. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.

Rationale for test conditions:

The experimental methods were conducted according to the methods described by Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and according to the relevant SOPs of the testing laboratory. .

Evaluation criteria:

The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in all Salmonella typhimurium and Escherichia coli WP2 uvrA bacterial strains
According to the guidelines [5][6][7][8], statistical method 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 item is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Statistics:

The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; 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 ExcelTM software.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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:

cytotoxicity

Remarks:

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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 (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
Precipitate/slight precipitate was detected on the plates in both Salmonella typhimurium strains with and without metabolic activation at 5000, 2500 and 1000 μg/plate concentrations in the preliminary experiment.
No inhibitory or toxic effects of the test item were detected in the preliminary experiment.

MUTAGENICITY TESTS (ASSAY 1 AND ASSAY 2)
In the Assay 1, the plate incorporation method was used. In the Assay 2, in case of all examined Salmonella typhimurium strains without metabolic activation the plate incorporation method was used. In case of all examined Salmonella typhimurium strains with metabolic activation and Escherichia coli WP2 uvrA with and without metabolic activation the pre-incubation method was used. The Assay 1, 2 were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The Assays were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1, 2 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Precipitate/slight precipitate was detected on the plates in all examined bacterial strains with and without metabolic activation at 5000 and/or 1581 μg/plate concentrations in Assays 1 and 2.
Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.19). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
In Assay 2 (plate incorporation method/pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.38). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

VALIDITY OF THE TESTS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.
The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Any other information on results incl. tables

Summary Tables of the Results

 

Summary Table of the Range Finding Test

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains
		TA98		TA100
		-S9	+S9	-S9	+S9
Untreated control	Mean	14.7	23.3	92.0	96.7
	MF	0.88	1.27	1.03	0.97
DMSO control	Mean	14.0	22.7	--	94.3
	MF	0.84	1.24	--	0.95
Distilled water control	Mean	--	--	88.3	--
	MF	--	--	0.99	--
PG + 2% Polysorbate 80 control	Mean	16.7	18.3	89.3	99.7
	MF	1.00	1.00	1.00	1.00
5000	Mean	13.7	23.3	98.3	94.0
	MF	0.82	1.27	1.10	0.94
2500	Mean	16.0	19.0	96.3	92.3
	MF	0.96	1.04	1.08	0.93
1000	Mean	14.0	14.7	91.0	95.7
	MF	0.84	0.80	1.02	0.96
316	Mean	12.0	23.0	100.7	98.3
	MF	0.72	1.25	1.13	0.99
100	Mean	20.7	22.3	94.0	101.0
	MF	1.24	1.22	1.05	1.01
31.6	Mean	18.3	20.3	95.7	101.7
	MF	1.10	1.11	1.07	1.02
10	Mean	17.7	18.7	92.0	96.3
	MF	1.06	1.02	1.03	0.97
NDP (4µg)	Mean	400.0	--	--	--
	MF	28.57	--	--	--
2AA (2µg)	Mean	--	2470.7	--	2469.3
	MF		109.00	--	26.18
SAZ (2µg)	Mean	--	--	1060.0	--
	MF	--	--	12.00	--

 

Summary Table of the Assay 1

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	18.3	21.0	94.7	106.0	12.7	11.0	8.0	7.3	43.3	47.3
	MF	0.98	1.02	1.03	1.14	0.79	1.00	1.09	1.05	0.98	0.97
DMSO control	Mean	18.3	22.3	--	105.7	--	12.7	7.7	7.0	--	47.7
	MF	0.98	1.08	--	1.13	--	1.15	1.05	1.00	--	0.98
Distilled water control	Mean	--	--	92.0	--	13.7	--	--	--	42.0	--
	MF	--	--	1.00	--	0.85	--	--	--	0.95	--
PG + 2% Polysorbate 80 control	Mean	18.7	20.7	92.0	93.3	16.0	11.0	7.3	7.0	44.0	48.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	17.0	21.0	84.0	87.3	11.0	12.7	7.3	7.3	40.0	45.7
	MF	0.91	1.02	0.91	0.94	0.69	1.15	1.00	1.05	0.91	0.94
1581	Mean	17.3	20.3	93.3	99.3	11.3	9.7	7.3	7.7	42.0	49.3
	MF	0.93	0.98	1.01	1.06	0.71	0.88	1.00	1.10	0.95	1.01
500	Mean	17.0	22.3	77.3	93.0	12.3	10.7	6.3	7.7	41.3	46.7
	MF	0.91	1.08	0.84	1.00	0.77	0.97	0.86	1.10	0.94	0.96
158.1	Mean	19.0	19.7	80.7	93.0	10.0	12.3	7.0	7.7	39.3	46.3
	MF	1.02	0.95	0.88	1.00	0.63	1.12	0.95	1.10	0.89	0.95
50	Mean	17.3	19.3	78.7	90.3	10.7	10.0	7.7	7.3	41.3	49.0
	MF	0.93	0.94	0.86	1.00	0.67	0.91	1.05	1.05	0.94	1.01
15.81	Mean	17.7	18.0	85.3	87.0	13.0	10.0	8.0	8.3	41.7	47.3
	MF	0.95	0.87	0.93	0.93	0.81	0.91	1.09	1.19	0.95	0.97
NPD (4µg)	Mean	410.7	--	--	--	--	--	--	--	--	--
	MF	22.40	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2408.0	--	2452.0	--	257.0	--	233.7	--	--
	MF	--	107.82	--	23.21	--	20.29	--	33.38	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	274.3
	MF	--	--	--	--	--	--	--	--	--	5.76
SAZ (2µg)	Mean	--	--	1253.3	--	1290.7	--	--	--	--	--
	MF	--	--	13.62	--	94.44	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	417.3	--	--	--
	MF	--	--	--	--	--	--	54.43	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1084.0	--
	MF	--	--	--	--	--	--	--	--	25.81	--

 

Summary Table of the Assay 2

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	17.3	23.3	95.3	104.3	15.3	15.3	7.7	9.0	54.0	57.7
	MF	0.90	1.00	0.96	1.05	1.05	1.05	0.96	1.29	1.09	1.01
DMSO control	Mean	18.0	21.7	--	98.7	--	13.3	8.7	8.0	--	57.0
	MF	0.93	0.93	--	0.99	--	0.91	1.08	1.14	--	0.99
Distilled water control	Mean	--	--	96.3	--	14.7	--	--	--	49.7	--
	MF	--	--	0.97	--	1.00	--	--	--	1.00	--
PG + 2% Polysorbate 80 control	Mean	19.3	23.3	99.0	99.7	14.7	14.7	8.0	7.10	49.7	57.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	19.3	20.3	95.3	108.3	16.0	15.0	9.3	6.7	53.3	57.7
	MF	1.00	0.87	0.96	1.09	1.09	1.02	1.17	0.95	1.07	1.01
1581	Mean	21.7	24.3	95.3	105.0	16.0	15.3	6.7	8.0	56.7	55.3
	MF	1.12	1.04	0.96	1.05	1.09	1.05	0.83	1.14	1.14	0.97
500	Mean	19.7	19.7	92.7	94.0	17.3	14.7	10.3	6.0	53.7	56.3
	MF	1.02	0.84	0.94	.094	1.18	1.00	1.29	0.86	1.08	0.98
158.1	Mean	19.0	25.0	99.0	96.7	16.7	16.0	9.7	6.7	48.7	59.0
	MF	0.98	1.07	1.00	0.97	1.14	1.09	1.21	0.95	0.98	1.03
50	Mean	19.3	27.3	104.7	96.0	17.0	15.7	7.0	7.0	53.7	58.0
	MF	1.00	1.17	1.06	0.96	1.16	1.07	0.88	1.00	1.08	1.01
15.81	Mean	19.0	22.0	95.3	102.0	14.7	16.0	9.0	9.7	48.7	57.7
	MF	0.98	0.94	0.96	1.02	1.00	1.09	1.13	1.38	0.98	1.01
NPD (4µg)	Mean	449.3	--	--	--	--	--	--	--	--	--
	MF	24.96	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2466.7	--	2432.0	--	233.7	--	210.0	--	--
	MF	--	113.85	--	24.65	--	17.53	--	26.25	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	258.3
	MF	--	--	--	--	--	--	--	--	--	4.53
SAZ (2µg)	Mean	--	--	1201.3	--	1210.7	--	--	--	--	--
	MF	--	--	12.47	--	82.55	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	448.0	--	--	--
	MF	--	--	--	--	--	--	51.69	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1108.0	--
	MF	--	--	--	--	--	--	--	--	22.31	--

 

Historical Control Data

(Period of 2011-2018)

Untreated control data
	Without metabolic activation (-S9)					With metabolic activation (+S9)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	T1535	TA1537	E. coli
Mean	22.3	101.5	12.1	7.9	36.3	27.4	108.9	11.6	9.4	41.3
St. dev.	5.4	19.5	4.6	3.5	10.8	6.7	18.4	3.6	3.8	10.3
Range	9-50	54-210	1-46	1-26	11-82	10-56	65-204	1-39	1-29	16-89
n	1860	1846	1857	1866	1875	1878	1869	1877	1881	1875
DMSO control data
	Without metabolic activation (-S9)					With metabolic activation (+S9)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	21.4	97.5	12.1	7.7	35.3	27.6	106.4	11.4	9.1	40.4
St. Dev.	5.3	18.8	4.5	3.4	10.7	6.7	19.3	3.9	3.7	10.2
Range	6-55	40-217	1-43	1-27	7-81	11-67	53-229	2-33	1-29	9-85
n	2007	1992	2004	2016	2019	2024	2013	2027	2028	2022
Distilled water control data
	Without metabolic activation (-S9)					With metabolic activation (+S9)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	23.0	101.0	12.1	8.7	37.4	29.0	108.9	11.4	10.0	42.3
St. Dev.	5.5	20.3	4.4	3.5	10.7	6.7	20.3	3.4	3.7	10.1
Range	11-45	45-215	2-47	2-24	12-84	10-53	64-222	3-39	1-24	13-91
n	399	1863	1869	405	1905	402	1875	1887	402	1893
DMF control data
	Without metabolic activation (-S9)					With metabolic activation (+S9)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.4	90.2	11.4	7.7	37.3	27.1	98.9	11.1	8.9	40.0
St. Dev.	5.2	16.8	4.3	3.4	12.8	6.8	18.0	3.3	3.5	11.0
Range	8-38	54-152	1-34	1-19	16-99	11-49	60-156	3-21	1-23	17-76
n	276	276	276	276	267	276	276	276	273	267
Acetone control data
	Without metabolic activation (-S9)					With metabolic activation (+S9)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.4	97.7	12.1	7.5	36.2	28.5	106.8	11.1	8.8	41.3
St. Dev.	5.0	14.7	5.6	2.9	9.6	6.6	14.2	3.4	3.3	9.1
Range	11-39	62-160	4-49	1-17	17-63	15-52	66-177	4-22	1-19	17-70
n	314	315	315	318	312	315	315	318	318	315
Positive reference control data
	Without metabolic activation (-S9)					With metabolic                activation (+S9)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	368.8	1208.5	1165.0	444.3	1034.2	2410.8	2425.1	229.2	219.0	255.4
St. Dev.	100.9	185.0	179.4	147.1	140.2	274.5	252.0	117.0	49.0	98.2
Range	152-2336	536-2120	208-2440	149-2104	488-2496	312-4918	1192-5240	101-2216	117-838	125-2512
n	1860	1848	1857	1866	1878	1878	1869	1881	1881	1875

TA98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella typhimurium TA1537, E. coli: Escherichia coli WPR uvrA, n: number of cases

Applicant's summary and conclusion

Conclusions:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The 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 metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, Assay 1 (Plate Incorporation Method) and Assay 2 (Plate Incorporation Method or Pre-Incubation Method).
The reported data of this mutagenicity assay show that 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.
In conclusion, the test item SynNova Base Oil (Batch Number: TS20371) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The 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-naphthoflavone-induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Plate Incorporation Method), Assay 1 (Plate Incorporation Method) and Assay 2 (Plate Incorporation Method or Pre-Incubation Method).

 

Based on the results of the Compatibility Test, the test item was dissolved in Propylene Glycol + 2% Polysorbate 80 at a maximum concentration of 100 mg/mL. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the absence and presence of metabolic activation were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate in Assays 1 and 2.

 

In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

 

Precipitate/slight precipitate was detected on the plates in all examined bacterial strains with and without metabolic activation at 5000 and/or 1581 μg/plate concentrations in Assays 1-2.

 

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.

 

The mean values of revertant colonies of the negative (vehicle/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 examined concentration range was considered to be adequate. The study was considered to be valid.

 

The reported data of this mutagenicity assay show that 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.

 

In conclusion, the test item SynNova Base Oil (Batch Number: TS20371) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.