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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January 2017 - 13 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO/IEC 17025:2005 (ISO/IEC, 2005)
Version / remarks:
2005
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Synonyms: p-tert-butylstyrene
CAS No: 1746-23-2
Purity: 95.9%

Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 98
Remarks:
reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens.
Species / strain / cell type:
S. typhimurium TA 100
Remarks:
reverted by mutagens that cause both frameshift and basepair substitution mutations.
Species / strain / cell type:
S. typhimurium TA 1535
Remarks:
reverted by mutagens that cause basepair substitutions
Species / strain / cell type:
S. typhimurium TA 1537
Remarks:
reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens.
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
sensitive to basepair substitution mutations, rather than frameshift mutations
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate (for tester strains TA98, TA1537, WP2 uvrA in the presence and absence of S9 activation and TA100, TA1535 in the presence of S9 activation); and
1.00, 3.33, 10.0, 33.3, 100, 333 and 1000 μg per plate (for tester strains TA100, TA1535 in the absence of S9 activation)
Vehicle / solvent:
DMSO (vehicle for Positive Controls: DMSO, except sterile water for sodium azide)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Initial cell density: 0.8 - 2 x 10(9) cells per mL


NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY : The preliminary toxicity assay was used to establish the dose-range over which the test substance would be assayed. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone and ten dose levels of the test substance, with a single plate/condition, on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. Dose levels for the mutagenicity assay were based upon post-treatment toxicity.

MUTAGENICITY ASSAY: One-half (0.5) milliliter of S9 or Sham mix (100 mM phosphate buffer at pH 7.4), 100 μL of tester strain (cells seeded) and 50.0 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test substance aliquot was replaced by a 50.0 μL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Evaluation criteria:
- For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.

- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0 times the mean vehicle control value and above the corresponding acceptable vehicle control range.

- Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0 times the mean vehicle control value and above the corresponding acceptable vehicle control range.

- An equivocal response is an increase in a revertant count that is greater than the acceptable vehicle control range but lacks a dose response or does not achieve the respective fold increase threshold cited.

- A response will be evaluated as negative, if it is neither positive nor equivocal.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 3333 μg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 3333 μg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 100 μg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 1000 μg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 100 μg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
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:
In preliminary toxicity assay, toxicity was observed beginning at 100, 667 or 3333 μg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate for tester strains TA98, TA1537, WP2 uvrA in the presence and absence of S9 activation and TA100, TA1535 in the presence of S9 activation and 1000 μg per plate for tester strains TA100, TA1535 in the absence of S9 activation.
Conclusions:
Under the study conditions, the test substance was determined to be non-genotoxic with or without metabolic activation in the Ames test.
Executive summary:

A study was conducted to determine the genotoxic potential of the test substanceaccording to OECD Guideline 471, in compliance with GLP.Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrA were treated with the test substance using both the Ames plate incorporation and preincubation methods, in triplicate both with and without the addition of a rat liver homogenate metabolizing system:Aroclor-induced rat liver (S9).In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 100, 667 or 3333 μg per plate. Based upon these results, in the mutagenicity assay, the dose levels tested were10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate for tester strains TA98, TA1537, WP2uvrA in the presence and absence of S9 activation and TA100, TA1535 in the presence of S9 activation and 1.00, 3.33, 10.0, 33.3, 100, 333 and 1000 μg per plate for tester strains TA100, TA1535 in the absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 100, 1000 or 3333 μg per plate with a few conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test (Dakoulas, 2018).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 2017 - 24 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
yes
Remarks:
The Study Director determines no impact to the study.
GLP compliance:
yes
Remarks:
Refer to main study report
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
CAS No.: 1746-23-2
Identification: para-tertiary-butyl styrene; TBS
Storage: 2 to 8°C, protected from light
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity assay: 6.25, 12.5, 25.0, 50.1, 100, 200, 401, 802 and 1603 μg/mL.
The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle.
Definitive mutagenicity assay: (A) 7, 14, 28.1, 37.5 and 50 μg/mL (4-hour treatment with S9) (B) 3.5, 7, 14, 28.1, 37.5 and 50 μg/mL (4-hour treatment without S9) (C) 3.5, 7, 14, 28.1, 37.5 and 50 μg/mL (24-hour treatment without S9)
Vehicle / solvent:
Test substance
- Vehicle(s)/solvent(s) used: DMSO for the test substance.
- Justification for choice of solvent/vehicle: Para-tert-butylstyrene (TBS) is soluble in dimethyl sulfoxide (DMSO) up to 500 mg/mL

Positive control substance
Methyl methanesulfonate (MMS) diluted in water for for the mutagenicity assay without S9. 7,12-Dimethyl-benz(a)anthracene (DMBA) was diluted in DMSO for the mutagenicity assay, with S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
Method of application: in medium

Duration
- Exposure duration: Target cells were treated with the test and control articles for 4 hours with and without S9, and for 24 hours without S9.
- Expression time (cells in growth medium): 2 days post-treatment
- Selection time (if incubation with a selection agent): 10-14 days

Selection agent (mutation assays): trifluorothymidine

Number of replications: Vehicle control in duplicate cultures and test article in single cultures in the preliminary toxicity assay. Duplicate cultures for the test and control substances in the definitive assay.

Number of cells evaluated: 3 x 10^6 cells/culture

Determination of cytotoxicity
- Method: relative total growth
Statistics:
None applied
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Relative total growth of the cloned cultures ranged from 28 to 92% (4-hour treatment with S9), 20 to 111% (4-hour treatment without S9) and 23 to 114% (24-hour treatment without S9).
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Test-specific confounding factors
- Effects of pH:
Preliminary toxicity assay: The test substance did not have an adverse impact on the pH of the cultures (pH 7.0 at the top dose).
Definitive Mutagenicity Assay: The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).

- Effects of osmolality:
Preliminary toxicity assay: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%.

- Precipitation:
Preliminary toxicity assay: Visible precipitate was observed at concentrations ≥100 μg/mL at the beginning of treatment. No visible precipitate was observed at the end of treatment.
Definitive Mutagenicity Assay: No visible precipitate was observed at the beginning or end of treatment

Results and discussion

Preliminary Toxicity Assay

For the results of the preliminary toxicity assay, kindly refer the attached background materials section of the IUCLID. para-tert-butylstyrene (TBS) was evaluated at concentrations of 6.25, 12.5, 25.0, 50.1, 100, 200, 401, 802 and 1603 µg/mL. The test substance formed clear solutions in DMSO at concentrations of 0.625 to 160.3 mg/mL. Visible precipitate was observed at concentrations ≥100 µg/mL at the beginning of treatment. No visible precipitate was observed at the end of treatment. The following observations were made while evaluating precipitation:

Dose Level (µg/mL)

Comments for 4

hour (-S9)

Comments for 4

hour (+S9)

Comments for 24

hour (-S9)

1603

Reddish-pink color

Reddish-pink color, 1 large irregularly shaped clump

Reddish-pink color, 1 irregular clump

802

1 large irregularly

shaped clump

Reddish-pink color, 1

irregular clump

401

1 large    irregularly

shaped clump

Reddish-pink color, 1 irregular clump

200

1 medium sized irregular clump

Reddish-pink color, 1 irregularly shaped

clump

100

-

Reddish-pink color, 1 irregular clump

50.1

-

-

Reddish-pink color, 1 irregularly shaped clump

25.0

-

-

-

12.5

-

-

-

6.25

-

-

-

The osmolality of the cultures was measured as follows:

Concentration Tested

Concentration (µg/mL)

Osmolality (mmol/kg)

Vehicle control

0

456

Highest

1603

423

Lowest precipitating

100

453

Highest soluble

50.1

454

The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%. The test substance did not have an adverse impact on the pH of the cultures (pH 7.0 at the top dose). Relative suspension growth (RSG) was 11, 54 and 100% at concentrations of 50.1 µg/mL (4- hour treatment with S9), 25 µg/mL (4-hour treatment without S9) and 25 µg/mL (24-hour treatment without S9), respectively. RSG was 0% at all higher concentrations using all treatment conditions.

Definitive Mutagenicity Assay

For the results of the definitive mutagenicity assays and Colony size distributions for the positive and vehicle control cultures, kindly refer the attached background materials section of the IUCLID. Based upon the results of the preliminary toxicity assay, the concentrations selected for the definitive mutagenicity assay were as indicated in the following table:

 

Treatment Condition

 

Treatment Time

 

 

Concentrations (µg/mL)

Non-activated

4 hours

3.5, 7, 14, 28.1, 37.5 and 50

24 hours

S9-activated

4 hours

7, 14, 28.1, 37.5 and 50

The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose). Cultures treated at concentrations of 7, 14, 28.1, 37.5 and 50 µg/mL (4-hour treatment with S9), 3.5, 7, 14 and 28.1 µg/mL (4-hour treatment without S9) and  3.5, 7, 14 and 28.1 µg/mL (24-

hour treatment without S9) exhibited 34 to 91%, 22 to 98% and 26 to 113% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 28 to 92% (4-hour treatment with S9), 20 to 111% (4-hour treatment without S9) and 23 to 114% (24-hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/106  clonable cells were observed under any treatment condition.

Trifluorothymidine-resistant colonies for the positive and vehicle control cultures, were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Conclusion

Under the conditions of the assay described in this report, para-tert-butylstyrene (TBS) was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Conclusions:
Under the study conditions, the test substance was concluded to be negative for the induction of forward mutations with and without metabolic activation, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Executive summary:

The test substance was evaluated for its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system according to OECD Guideline 490, in compliance with GLP. Dimethyl sulfoxide (DMSO) was used as the vehicle. In the preliminary toxicity assay, the concentrations tested were 6.25, 12.5, 25.0, 50.1, 100, 200, 401, 802 and 1603 μg/mL. The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle. Visible precipitate was observed at concentrations ≥100 μg/mL at the beginning of treatment. No visible precipitate was observed at the end of treatment. Relative suspension growth (RSG) was 11, 54 and 100% at concentrations of 50.1 μg/mL (4 h treatment with S9), 25 μg/mL (4 h treatment without S9) and 25 μg/mL (24 h treatment without S9), respectively. RSG was 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 7, 14, 28.1, 37.5 and 50 μg/mL (4 h treatment with S9), 3.5, 7, 14, 28.1, 37.5 and 50 μg/mL (4 h treatment without S9) and 3.5, 7, 14, 28.1, 37.5 and 50 μg/mL (24 h treatment without S9). In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 7, 14, 28.1, 37.5 and 50 μg/mL (4 h treatment with S9), 3.5, 7, 14 and 28.1 μg/mL (4 h treatment without S9) and 3.5, 7, 14 and 28.1 μg/mL (24 h treatment without S9) exhibited 34 to 91%, 22 to 98% and 26 to 113% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 28 to 92% (4 h treatment with S9), 20 to 111% (4 h treatment without S9) and 23 to 114% (24 h treatment without S9). No increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed under any treatment condition. The study fufilled all validity criteria. Under the study conditions, the test substance was concluded to be non-mutagenic in the in vitro L5178Y/TK+/- mouse lymphoma assay (Dutta, 2018).

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March 2017 - 13 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
updated and adopted 26 September 2014
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Synonyms: p-tert-butylstyrene
CAS No: 1746-23-2
Purity: 95.9%
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
For lymphocytes:
- Sex, age and number of blood donors: Male, Age: 33
- Whether blood from different donors were pooled or not: No

The donor had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals (Clare et al., 2006).

Cytokinesis block (if used):
Cytochalasin B (cytoB) was dissolved in DMSO and used at 6 μg/mL concentration to block cytokinesis. The cyto B exposure time for the 4 h treatment in the non-activated and the S9-activated studies was 20 h (± 30 minutes).
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3717, Exp. Date: 09 Nov 2018) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared on the day of use at below final concentrations in culture medium*:
NADP (sodium salt): 1 mM
Glucose-6-phosphate: 1 mM
Potassium chloride: 6 mM
Magnesium chloride: 2 mM
S9 homogenate: 20 μL/mL
*RPMI 1640 Serum-free medium supplemented with 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin
Test concentrations with justification for top dose:
Based on the results of the preliminary toxicity test, the doses selected for testing in the micronucleus assay were as follows:
Non-activated (4 h): 5, 15, 20, 25, 30, 35, 40, 45, 50 µg/mL
Non-activated (24 h): 5, 15, 20, 25, 30, 35, 40, 45, 50 µg/mL
S9-activated (4 h): 5, 15, 25, 50, 75, 100, 125, 150 µg/mL
Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the micronuclei was selected based on 55 ± 5% cytotoxicity (CBPI relative to the vehicle control) at all testing conditions (i.e., 4 h activated and non-activated and 24 h non-activated)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the information provided by the Sponsor, the solubility of the test substance, and compatibility with the target cells. Para-tert-butylstyrene (TBS) was soluble in dimethyl sulfoxide (DMSO) up to 500 mg/mL (as confirmed in BioReliance Study No. AE80EB.502REACH.BTL).
- The vehicle and positive controls have been characterized as per the Certificates of Analysis on file. The stability of the vehicle and positive controls and their mixtures was demonstrated by acceptable results that met the criteria for a valid test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
vinblastine
Remarks:
- Vinblastine (for -S9 test system) was used at 5, 7.5, and 10 ng/mL concentrations in sterile water - Cyclophosphamide (for + S9 test system) was used at 2.5, 5 and 7.5 μg/mL concentrations in sterile water
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

FREQUENCY AND ROUTE OF ADMINISTRATION:
Target cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9, by incorporation of the test substance vehicle mixture into the treatment medium.

PRELIMINARY TOXICITY TEST FOR SELECTION OF DOSE LEVELS:
- Human peripheral blood lymphocytes (HPBL) were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, lowest precipitating dose, and the highest soluble dose was measured. Dose levels for the micronucleus assay were based upon post-treatment toxicity (CBPI relative to the vehicle control).
- Test concentrations: The doses tested ranged from 0.16 to 1600 μg/mL (10 mM), which was the limit dose for this assay.

TREATMENT OF TARGET CELLS:
- Preparation of Target Cells: Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin, 100 μg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.
- pH: The pH of the highest dose of dosing solution in the treatment medium was measured using test tape.
- Volumes: 5 mL of complete medium and 4 mL of serum free medium were added to non-activated and activated test systems respectively; 50 μL of vehicle or test substance dosing solution; 1 mL of S9 mix
After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytoB at 6.0 μg/mL and returned to the incubator under standard conditions. For the 24 hour treatment in the non-activated study, cytoB (6.0 μg/mL) was added at the beginning of the treatment.

COLLECTION OF CELLS:
Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986).
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and were either stored overnight or longer at 2-8°C, or the slides were prepared immediately after harvest. To prepare slides, the cells were collected by centrifugation and when necessary, the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange and identified by the BioReliance study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.

CELL CYCLE KINETICS SCORING:
For the preliminary toxicity test, at least 500 cells, if possible, were evaluated to determine the CBPI at each dose level and the control.
For the micronucleus assay, at least 1,000 cells (500 cells per culture), if possible, were evaluated to determine the CBPI at each dose level and the control.
The CBPI was determined using the following formula:
CBPI = (1X Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells)/Total number of cells scored
% Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)}
t = test substance treatment culture
c = vehicle control culture

MICRONUCLEUS SCORING:
The slides from at least three test substance treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei. Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
- the micronucleus should have the same staining characteristics as the main nucleus
- the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
- the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus

CRITERIA FOR DETERMINATION OF A VALID TEST:
Vehicle Controls: The frequency of cells with micronuclei should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are not extreme outliers (indicative of experimental or human error).
Positive Controls: The percentage of micronucleated cells must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).
Cell Proliferation: The CBPI of the vehicle control at harvest must be ≥ 1.4.
Test Conditions: The test substance must be tested using a 4-hour treatment with and without S9, as well as a 24-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.
Analyzable Concentrations: At least 2000 binucleated cells from at least three appropriate test substance concentrations.
Evaluation criteria:
The test substance was considered to have induced a positive response if:
- at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
- the increase was concentration-related (p ≤ 0.05), and
- results were outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary test results:
- Cytotoxicity (≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) was observed at doses ≥ 48 μg/mL in the non-activated 4 and 24-h exposure groups, and at doses ≥ 160 μg/mL in the S9-activated 4-h exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 480 μg/mL in all three treatment conditions. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 50 μg/mL for the non-activated 4 and 24-h exposure groups; and from 5 to 150 μg/mL for the S9-activated 4-h exposure group.
- The osmolality of the test substance doses in treatment medium was considered acceptable. The pH of the highest dose of test substance in treatment medium was 7.5.

Micronucleus assay results:
- The pH of the highest dose of test substance in treatment medium was 7.5.
- Cytotoxicity (≥ 50% CBPI relative to the vehicle control) was observed at doses ≥ 45 μg/mL in the non-activated 4-h exposure group; at doses ≥ 75 μg/mL in the S9-activated 4-h exposure group; and at doses ≥ 35 μg/mL in the non-activated 24-h exposure group. The doses selected for evaluation of micronuclei were 15, 30, and 45 μg/mL for the non-activated 4-h exposure group; 5, 25, and 75 μg/mL for the S9-activated 4-h exposure group; and 5, 20, and 35 μg/mL for the non-activated 24-h exposure group.
- No significant or dose-dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).
- The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.
Remarks on result:
other: non-clastogenic

Table 1: Micronucleus analysis of human peripheral blood lymphocytes treated with para-tert-butylstyrene (TBS) in the absence of exogenous metabolic activation

Definitive assay: 4-h treatment, 24-h harvest
Treatment Replicate Total # Percentage of Average Percent
μg/mL Culture of Binucleated Micronucleated Micronucleated
Cells Binucleated Cells Binucleated
Counted per culture Cells per Dose
DMSO A 1000 0.5 0.4
B 1000 0.3
TBS
15 A 1000 0.5 0.5
B 1000 0.5
30 A 1000 0.4 0.35
B 1000 0.3
45 A 1000 0.3 0.45
B 1000 0.6

Table 2: Micronucleus analysis of human peripheral blood lymphocytes treated with para-tert-butylstyrene (TBS) in the presence of exogenous metabolic activation

Definitive assay: 4-h treatment, 24-h harvest
Treatment Replicate Total # Percentage of Average Percent
μg/mL Culture of Binucleated Micronucleated Micronucleated
Cells Binucleated Cells Binucleated
Counted per culture Cells per Dose
DMSO A 1000 0.3 0.35
B 1000 0.4
TBS
5 A 1000 0.3 0.3
B 1000 0.3
25 A 1000 0.3 0.4
B 1000 0.5
75 A 1000 0.3 0.3
B 1000 0.3
CP, 5 A 1000 1.1 1.10**
B 1000 1.1
** p ≤ 0.01, Fisher's exact test, relative to the solvent control.

Table 3: Micronucleus analysis of human peripheral blood lymphocytes treated with para-tert-butylstyrene (TBS) in the absence of exogenous metabolic activation

Definitive assay: 24-h treatment, 24-h harvest
Treatment Replicate Total # Percentage of Average Percent
μg/mL Culture of Binucleated Micronucleated Micronucleated
Cells Binucleated Cells Binucleated
Counted per culture Cells per Dose
DMSO A 1000 0.3 0.3
B 1000 0.3
TBS
5 A 1000 0.5 0.4
B 1000 0.3
25 A 1000 0.4 0.4
B 1000 0.4
75 A 1000 0.3 0.35
B 1000 0.4
CP, 5 A 1000 1.5 1.35**
B 1000 1.2
** p ≤ 0.01, Fisher's exact test, relative to the solvent control.
Conclusions:
Under the study conditions, the test substance was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems.
Executive summary:

A study was conducted to determine the genotoxic potential of test substance, para-tert-butylstyrene (TBS), using peripheral blood lymphocytes (HPBL), according to OECD Guideline 487 (in vitro mammalian cell micronucleus test), in compliance with GLP. In a preliminary toxicity assay, HPBL were exposed to vehicle alone and to 9 concentrations of test substance with half-log dose spacing using single cultures in the absence and presence of Aroclor-induced rat liver (S9) metabolic activation. Dimethyl sulfoxide (DMSO) was used as the vehicle. The doses tested ranged from 0.16 to 1600 μg/mL (10 mM). Precipitation of test substance in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. Cytotoxicity (≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) was observed at doses ≥48 μg/mL in the non-activated 4 and 24 h exposure groups, and at doses ≥160 μg/mL in the S9 activated 4 h exposure group. Also, visible precipitate was observed at doses ≥480 μg/mL in all three treatment conditions. Based on the results, the doses chosen for the main micronucleus assay ranged from 5 to 50 μg/mL for the non-activated 4 and 24 h exposure groups; and from 5 to 150 μg/mL for the S9 activated 4 h exposure. Greater than 50% cytotoxicity was observed at doses ≥45 μg/mL in the non-activated 4 h exposure group; at doses ≥75 μg/mL in the S9 activated 4 h exposure group; and at doses ≥35 μg/mL in the non-activated 24-h exposure group. The doses selected for evaluation of micronuclei were 15, 30, and 45 μg/mL for the non-activated 4 h exposure group; 5, 25, and 75 μg/mL for the S9 activated 4 h exposure group; and 5, 20, and 35 μg/mL for the non-activated 24 h exposure group. No significant or dose-dependent increases in micronuclei induction were observed in treatment groups with or without S9. Under the study conditions, the test substance was determined to be non-clastogenic with or without metabolic activation in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes (Roy, 2019).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation study in bacteria

A study was conducted to determine the genotoxic potential of the test substance according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation and preincubation methods, in triplicate both with and without the addition of a rat liver homogenate metabolizing system: Aroclor-induced rat liver (S9). In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 100, 667 or 3333 μg per plate. Based upon these results, in the mutagenicity assay, the dose levels tested were 10.0, 33.3, 100, 333, 1000, 3333 and 5000 μg per plate for tester strains TA98, TA1537, WP2uvrA in the presence and absence of S9 activation and TA100, TA1535 in the presence of S9 activation and 1.00, 3.33, 10.0, 33.3, 100, 333 and 1000 μg per plate for tester strains TA100, TA1535 in the absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 100, 1000 or 3333 μg per plate with a few conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test (Dakoulas, 2018).

In vitro gene mutation study in mammalian cells

The test substance was evaluated for its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system according to OECD Guideline 490, in compliance with GLP. Dimethyl sulfoxide (DMSO) was used as the vehicle. In the preliminary toxicity assay, the concentrations tested were 6.25, 12.5, 25.0, 50.1, 100, 200, 401, 802 and 1603 μg/mL. The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle. Visible precipitate was observed at concentrations ≥100 μg/mL at the beginning of treatment. No visible precipitate was observed at the end of treatment. Relative suspension growth (RSG) was 11, 54 and 100% at concentrations of 50.1 μg/mL (4 h treatment with S9), 25 μg/mL (4 h treatment without S9) and 25 μg/mL (24 h treatment without S9), respectively. RSG was 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 7, 14, 28.1, 37.5 and 50 μg/mL (4 h treatment with S9), 3.5, 7, 14, 28.1, 37.5 and 50 μg/mL (4 h treatment without S9) and 3.5, 7, 14, 28.1, 37.5 and 50 μg/mL (24 h treatment without S9). In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 7, 14, 28.1, 37.5 and 50 μg/mL (4 h treatment with S9), 3.5, 7, 14 and 28.1 μg/mL (4 h treatment without S9) and 3.5, 7, 14 and 28.1 μg/mL (24 h treatment without S9) exhibited 34 to 91%, 22 to 98% and 26 to 113% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 28 to 92% (4 h treatment with S9), 20 to 111% (4 h treatment without S9) and 23 to 114% (24 h treatment without S9). No increases in induced mutant frequency ≥90 mutants/10E+6 clonable cells were observed under any treatment condition. The study fulfilled all validity criteria. Under the study conditions, the test substance was concluded to be non-mutagenic in the in vitro L5178Y/TK+/- mouse lymphoma assay (Dutta, 2018).

In vitro cytogenetic study in mammalian cells

A study was conducted to determine the genotoxic potential of test substance according to OECD Guideline 487 in compliance with GLP. In a preliminary toxicity assay, human peripheral blood lymphocytes were exposed to vehicle alone and to 9 concentrations of test substance with half-log dose spacing using single cultures in the absence and presence of Aroclor-induced rat liver (S9) metabolic activation. Dimethyl sulfoxide (DMSO) was used as the vehicle. The doses tested ranged from 0.16 to 1600 μg/mL (10 mM). Precipitation of test substance in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. Cytotoxicity (≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) was observed at doses ≥48 μg/mL in the non-activated 4 and 24 h exposure groups, and at doses ≥160 μg/mL in the S9 activated 4 h exposure group. Also, visible precipitate was observed at doses ≥480 μg/mL in all three treatment conditions. Based on the results, the doses chosen for the main micronucleus assay ranged from 5 to 50 μg/mL for the non-activated 4 and 24 h exposure groups; and from 5 to 150 μg/mL for the S9 activated 4 h exposure. Greater than 50% cytotoxicity was observed at doses ≥45 μg/mL in the non-activated 4 h exposure group; at doses ≥75 μg/mL in the S9 activated 4 h exposure group; and at doses ≥35 μg/mL in the non-activated 24-h exposure group. The doses selected for evaluation of micronuclei were 15, 30, and 45 μg/mL for the non-activated 4 h exposure group; 5, 25, and 75 μg/mL for the S9 activated 4 h exposure group; and 5, 20, and 35 μg/mL for the non-activated 24 h exposure group. No significant or dose-dependent increases in micronuclei induction were observed in treatment groups with or without S9. Under the study conditions, the test substance was determined to be non-clastogenic with or without metabolic activation in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes (Roy, 2019).

Justification for classification or non-classification

Based on the results of the in vitro genotoxicity studies in bacterial and mammalian cells, the test substance does not warrant classification according to EU CLP (1272/2008/EC) criteria.