Reaction mass of octan-2-ylbenzene, octan-3-ylbenzene and octan-4-ylbenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2019 - 23 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:

21 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:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(octan-2-yl)benzene

EC Number:

950-576-0

Cas Number:

31047-57-1

Molecular formula:

C14H22

IUPAC Name:

(octan-2-yl)benzene

Test material form:

liquid

Details on test material:

Identification: SHR 1396
Physical Description: Clear colorless liquid
Storage Conditions: At room temperature

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Remarks:

bacteria

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

bacteria

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight)
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP, and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The solution was filter-sterelized. 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.
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 mL S9-mix in 3mL top molten agar with 0.1 mL of a dilution of the test item in vehicle and 0.1 mL of fresh bacterial culture.
- quality controls of S9: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Test concentrations with justification for top dose:

Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate.
The highest concentration of SHR 1396 used in the subsequent mutation assays was 5000 μg/plate or the level at which the test item exhibited limited solubility.
Main study: TA1535, TA1537 and TA98 (without and with S9-mix): 52, 164, 512, 1600 and 5000 µg/plate

Experiment 2, TA1535, TA1537, TA100 and WP2uvrA:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2A, TA98:
Without S9 mix: 26, 82, 256, 800, 2500 µg/plate
With S9 mix: 26, 82, 256, 800, 2500, 5000 µg/plate

Vehicle / solvent:

- Solvent used for test item: Ethanol
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in ethanol at concentrations of 100 mg/mL and below.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Saline

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9: 5 µg/plate in saline for TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

without S9: 2.5 µg/plate in DMSO for TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9: 15 µg/plate in DMSO for TA1537 (pre-incubation assay)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9: 10 µg/plate in DMSO for TA98

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9: 650 µg/plate in DMSO for TA100

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9: 10 µg/plate in DMSO for WP2uvrA

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9: 2.5 µg/plate in DMSO for TA1535, TA 1537; 1 µg/plate in DMSO for TA98 and TA100 (direct plate assay); 5 µg/plate in DMSO for TA100 (pre-incubation assay); 15 µg/plate in DMSO for WP2uvrA.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^9 cells/mL
- Test substance added in agar (plate incorporation), preincubation

DOSE RANGE FINDING TEST
- Strains TA100 and WP2uvrA, with and without S9-mix.
- Concentrations: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate, tested in triplicate.

TREATMENT:
First experiment: Direct plate assay. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item 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.

Second experiment: Pre-incubation assay. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, 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), 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.05 mL of a dilution of the test item in ethanol. After the pre-incubation period the solutions were added to 3 mL molten top agar. 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 item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ENVIRONMENTAL CONDITIONS
- Incubation at 37.0 ± 1.0°C (actual range 35.4 - 38.1°C).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

ACCEPTABILITY 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 vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) 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.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

INTERPRETATION
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent 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 follow up experiment.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only in the second experiment, with and without S9 mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only in the second experiment, without S9 mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

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

Additional information on results:

PRECIPITATE
In the dose rage finding, precipitation of SHR 1396 on the plates was not observed at the end of the incubation period in any tester strain.
In the first experiment, precipitation of SHR 1396 on the plates was observed at 1600 and 5000 μg/plate at the end of the incubation period.
In the second experiment, precipitation of SHR 1396 on the plates was observed at the concentrations of 1600, 2500 and/or 5000 μg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES
In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

TOXICITY
In the first experiment, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA1537 (absence and presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reduction are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
In the second experiment cytotoxicity was observed in tester strains TA100 in the absence and presence of S9-mix, at 52 μg/plate and upwards and 512 μg/plate and upwards respectively, and TA1537 in the absence of S9-mix at the highest concentration of 5000 μg/plate.

RESULTS
- Individual plate counts : see 'detail tables' in attachment
- Mean number of revertant colonies per plate and standard deviation : see 'summary tables' in attachment

HISTORICAL CONTROL DATA: see tables included in 'any other information on results including tables'

Any other information on results incl. tables

Historical control data solvent control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 – 27	3 – 20	3 – 23	8 - 61	8 – 60	61 – 176	60 - 176	10 – 61	9 - 68
Mean	10	11	6	6	16	22	112	108	27	33
SD	3	3	2	3	5	7	18	21	8	9
n	3303	3265	3232	3212	3251	3326	3336	3246	3021	2993

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Apr 2016 and Apr 2019

Historical control data of the positive control items

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	128 – 1530	73 – 1481	58 – 1422	54 – 1239	365 – 1978	250 – 2018
Mean	919	256	802	328	1305	910
SD	172	122	362	154	236	355
n	3215	3122	2777	3187	3202	3216

 

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	439 – 1993	408 - 2379	93 – 1999	109 - 1968
Mean	907	1308	1073	437
SD	167	386	537	158
n	3231	3179	2923	2987

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Apr 2016 and Apr 2019. 

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD471 guideline and GLP principles, SHR 1396 was found not to be mutagenic with or without metabolic activation.

Executive summary:

An in vitro gene mutation (AMES) test was performed according to OECD 471 guideline and in accordance with GLP principles. All bacterial strains TA1535, TA1537, TA100, TA98 and WP2uvrA showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Cytotoxicity was observed in tester strains TA100 in the absence and presence of S9 mix and in the tester strain TA 1537 in the absence of S9 mix. No cytotoxicity and/or precipitation of the test substance was observed in the other tester strains. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that SHR 1396 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.