Isobutylbenzene

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:

19 Sep - 28 Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate produced by "Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland Pfalz"

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isobutylbenzene

EC Number:

208-706-2

EC Name:

Isobutylbenzene

Cas Number:

538-93-2

Molecular formula:

C10H14

IUPAC Name:

(2-methylpropyl)benzene

Test material form:

liquid

Details on test material:

- batch no.: EXP-18/11/346
- State of aggregation: liquid
- Log KOW: 3.94 (calculated); 4.8 at 23°C and pH 6.0 (measured)
- Molecular weight: 134.22 g/mol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:BASF SE; batch no. EXP-18/11/346
- Purity: 99.9 area-%
- Physical state: liquid
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed until Oct 2020
- Stability of the test substance in the solvent/vehicle: The stability of the test substance in the vehicle DMSO was not determined analytically, because the test substance was administered immediately after preparation and is usually stable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in dimethyl sulfoxide (DMSO). To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before administration.

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in dimethyl sulfoxide (DMSO)

Method

Target gene:

his, trp

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 (supplemented with cofactors) derived from phenobarbital (i.p.) and β-naphthoflavone (orally) induced rat liver

Test concentrations with justification for top dose:

In agreement with the recommendations of current guidelines 5 mg/plate were selected as maximum test dose.

Doses in the 1st Experiment (Standard plate test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate

Doses in the 2nd Experiment (Preincubation test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (TA 1535, TA 100, TA 98 and E. coli);
0; 3.3; 10; 33; 100; 333 and 1000 μg/plate (TA 1537); No mutagenicity was observed in the standard plate test. Due to toxicity, the doses were adjusted in the preincubation test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Controls

Untreated negative controls:

yes

Remarks:

sterility controls (plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene

Remarks:

additional positive controls: N-methyl-N'-nitro-N-nitrosoguanidine (5 μg/plate; dissolved in DMSO; strains: TA 1535, TA 100; without S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): approx. 10^8 cells/ml

CELLS USED
- Source of cells: Moltox Molecular Toxicology, Inc., Boone, NC 28607; USA
- Suitability of cells: The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid). E. coli WP2 uvrA was checked for UV sensitivity. Histidine and tryptophan auxotrophy was checked in each experiment via the spontaneous
rate.
- Methods for maintenance in cell culture: deep-frozen (-70°C to -80°C) bacterial cultures were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours (overnight). The optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL).

DURATION
- Preincubation period: about 20 minutes
- Exposure duration: 48 - 72 h

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6) and clearing or diminution of the background lawn (= reduced his- or trp- background growth)

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli
WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and
TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix
or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative
control data under all experimental conditions in at least two experiments carried out
independently of each other.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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 applicable

Positive controls validity:

valid

Test results 3

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 applicable

Positive controls validity:

valid

Test results 4

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 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the standard plate test, precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: see table 2
- Negative (solvent/vehicle) historical control data: see table 1

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- A bacteriotoxic effect was observed depending on the strain and test conditions from about 3.3 μg/plate onward (for details see table 3)

Any other information on results incl. tables

Tab. 1: Historical Negative Controls 

Strain S9 Mix		Positive	No. of	No. of	Min	Max	Mean	SD
		control	Plates	Values

TA 1535Without	(All)	414	152	7	16	10	2.0
With	(All)	414	152	6	18	10	2.0
TA 100Without	(All)	426	152	71	132	100	11.4
With	(All)	417	153	70	147	107	13.7
TA 1537Without	(All)	411	152	5	13	8	1.7
With	(All)	411	152	5	16	9	2.1
TA 98Without	(All)	420	152	14	34	21	3.3
With	(All)	413	152	12	38	28	4.5
E. coliWithout	(All)	396	152	15	34	24	3.9
With	(All)	399	152	17	36	25	4.4

Tab. 2: Historical Positive Controls 

Strain S9 Mix		Positive	No. of	No. of	Min	Max	Mean	SD
		control	Plates	Values

TA 1535Without	MNNG	312	110	1541	6171	3967	1253.9
With	2-AA	312	110	105	520	197	56.3
TA 100Without	MNNG	315	110	1126	5557	3298	1109.6
With	2-AA	309	111	272	3021	1748	587.2
TA 1537Without	AAC	309	110	253	2190	1044	404.3
With	2-AA	312	110	50	399	141	50.3
TA 98Without	NOPD	309	110	324	1746	863	206.8
With	2-AA	315	110	493	3096	1524	529.1
E. coliWithout	4-NQO	306	110	164	1721	860	431.9
With	2-AA	306	110	61	537	133	61.8

Tab. 3: Observed bacteriotoxicity

Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	Without	-	-	333 – 5000	1000 – 5000	-
	With	-	-	100 – 5000	5000	-
2nd-PIT	Without	333 – 5000	333 – 5000	3.3 – 10, 100 - 1000	100 – 5000	-
	With	2500 – 5000	1000 – 5000	1000	1000 – 5000	-

 - = no adverse effect observed

Reduced background growth was observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	Without	2500 – 5000	2500 – 5000	1000 – 5000	2500 – 5000	-
	With	2500 – 5000	2500 – 5000	1000 – 5000	2500 – 5000	-
2nd-PIT	Without	333 – 5000	333 – 5000	333 – 1000	333 – 5000	333 – 5000
	With	333 – 5000	333 – 5000	333 – 1000	333 – 5000	333 – 5000

 - = no adverse effect observed

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS:                     TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE:             33 μg - 5000 μg/plate (SPT)

3.3 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY:              In the standard plate test, precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix.

TOXICITY:                  A bacteriotoxic effect was observed depending on the strain and test conditions from about 3.3 μg/plate onward.

MUTAGENICITY:

A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.