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Genetic toxicity in vitro

Description of key information

GLP-studies equivalent to OECD guideline 471 are available for DVB-96 and DVB-HP. For DVB-55, non-GLP studies equivalent to OECD guideline 471 and 480 are available. This data is supported by a publication reporting Ames test results for an unknown grade of the reaction mass. In addition, a GLP-study equivalent to OECD guideline 473 is available for DVB-96. For DVB-55 and DVB-HP GLP-studies according to OECD guideline 476 are available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Non-GLP study equivalent to OECD guideline 471.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
TA1535- hisG46, TA1537- hisC3076, TA1538- hisD3052, TA98- hisD3052, TA100-hisG46
Species / strain / cell type:
other: S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100; yeast S. cerevisiae
Metabolic activation:
with and without
Metabolic activation system:
Livers from adult male mice induced with a polychlorinated biphenyl (Aroclor 1254)
Test concentrations with justification for top dose:
Bacteria - 0.5, 1, 5, 10, 50, 100 or 500 µg/plate
Yeast - 0.01 or 0.1 (w/v or v/v)
Vehicle / solvent:
not reported
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-o-tolylazo-o-toluidine and N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
S. typhimurium
The S. typhimurium strains used at SRI were obtained from Dr. Bruce Ames of the University of California at Berkeley. All are histidine auxotrophs (his-) by virtue of mutations in the histidine operon. All the indicator strains are stored at -80 C. For each experiment, an inoculum from frozen stock culture is grown overnight at 37°C in a nutrient broth consisting of 1% tryptone and 0.5% yeast extrract. After stationary overnight growth, the cultures are shaken for 3 to 4 hours to ensure optimal growth. Each culture is checked for sensitivity to crystal violet. The presence of the rfa- mutation makes the indicator + strains sensitive to this dye, whereas the parent strain, rfa , is not sensitive to the dye. However, the mutation is reversible, leading to the accumulation of rfa+ cells in the culture. Therefore, the cells must be tested routinely to ensure their sensitivity to crystal violet. Each culture also is tested by specific mutagens known to revert each test strain (positive controls) .

To a sterile 13 x 100 test tube placed in a 43°C heating block, add in the following order:
(1) 2 mL of 0.6% agar*
(2) 0.1 mL of indicator organism
(3) 0.15 mL of metabolic activation mixture (optional)
(4) Up to 100 µL of a solution of the test chemical

For negative controls, use steps 1, 2 , and 3 (optional) and use 100 µL of the solvent used for the test chemical. This mixture was stirred gently and then poured onto minimal agar plates. After the soft agar had set, the plates were incubated at 37°C for two days. The number of hisf revertants (colonies that grow on plates lacking a sufficient amount of histidine to support colony formation) were counted and recorded. Some of the revertants were routinely tested to be sure that they are his+, require biotin, and were sensitive to crystal violet (rfa).

Saccharomyces Cerevisiae D3: The yeast cerevisiae D3 is a diploid and is heterozygous for a mutation in an adenine-metabolizing enzyme. The Saccharomyces test strain from the liquid nitrogen was grown overnight at 30°C with aeration in 1.0% tryptone and 0.5% yeast extract. The cells were washed twice in 0.067 M PO4, buffer (pH 7.4) and resuspended in the same buffer at a concentration of 10E 8 cells/mL. The in vitro yeast mitotic recombination assay in suspension consists of 5E7 washed, stationary-phase yeast cells in 1 mL of 0.067 M PO4 buffer (pH 7.4) and 50 mg/mL of the test chemical (or a fraction of the concentration required to give 50% killing). The suspension was incubated at 30°C for 4 hours. After incubation, the sample was diluted serially in sterile saline and plated on tryptone yeast agar plates. Plates of a dilution were incubated for 2 days at 30°C, followed by 2 days at 4°C to enhance the development of red pigment that is indicative of adenine-negative homozygosity. Plates were screened for red colonies or red sectors by scanning the plates with a dissecting microscope at 10 x magnification. Plates of a dilution were incubated for 2 days at 30°C for determination of the total number of colony-forming units. The in vitro yeast mitotic recombination assay in suspension with metabolic activation was carried out as above with the addition of the metabolic activation system to the incubation mixture.

Metabolic Activation: Adult male mice were given a single intraperitoneal injection of a polychlorinated biphenyl (Aroclor 1254) at a dosage of 500 mglkg. Four days after the injection, the food was removed. On the fifth day, the mice were sacrificed. The livers were removed aseptically and placed in preweighed, sterile glass beakers. The organ weight was determined, and all subsequent operations to the metabolic activation step were conducted in an ice bath. The organ was washed in an equal volume of cold, sterile 0.15M KCl (1 mL/g of wet organ), minced with sterile surgical scissors in three volumes of 0.15 KCl, and homogenized with a Potter-Elvehjem apparatus. The homogenate was centrifuged for 10 minutes at 9000 x g, and the supernate was removed and stored in liquid nitrogen. To the postmitochondrial supernate were added MgCl2, KCl, glucose-6-phosphate, TPN, and sodium phosphate (pH 7.4).
Evaluation criteria:
No data
Statistics:
No data
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
Saccharomyces cerevisiae
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100; yeast S. cerevisiae
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Divinylbenzene was not mutagenic in the S. typhimurium assay and was negative in the mitotic recombination assay with S. cerevisiae D3.
Conclusions:
DVB-55 was not mutagenic in the S. typhimurium assay with and without activation and was negative in the mitotic recombination assay with S. cerevisiae D3.
Executive summary:

Seventeen compounds were examined for mutagenic activity with five strains of the bacteria Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538) and with the yeast Saccharomyces cerevisiae D3. Each assay was performed in the presence and in the absence of a metabolic activation system. None of the seventeen compounds were mutagenic in the bacterial assay with Salmonella typhimurium, either in the presence or in the absence of the liver metabolic activation system. One of the compounds, S-2 increased mitotic recombination in the Saccharomyces cerevisiae D3 assay, whereas S-4, S-5, and S-10 gave marginally positive responses. Compounds S-3, S-6, S-7, S-8, S-9, S-11 (divinylbenzene), S-12, S-13, and S-14 did not increase mitotic recombination. Assays S. cerevisiae with S-1, S-16, and S-17 were incomplete at the time of reporting.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 October 2009 to 23 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP-study according to OECD guideline 476
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
To assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes

- Periodically checked for Mycoplasma contamination:yes

- Periodically checked for karyotype stability: no

- Periodically "cleansed" against high spontaneous background: yes

Cell Line
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.
Cell Culture
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.
Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.



Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
PB/betaNF S9 was prepared from the livers of male Sprague Dawley CD strain rats. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 prepreparation on the fourth day.
Test concentrations with justification for top dose:
The test material was accurately weighed and dissolved in Dimethyl sulphoxide (DMSO) and appropriate dilutions made. The test material was considered to be a complex mixture and therefore the maximum dose level was 5000 µg/mL, the maximum recommended dose level.
Vehicle and positive controls were used in parallel with the test material. Solvent treatment groups were used as the negative controls and Ethyl methane sulphonate (EMS) Sigma Batch Number 142314732109252 at 500 and 750 µg/mL was used as the positive control in cultures without S9. Dimethyl benzanthracene (DMBA) Sigma Batch Numbers 024K0757 and 105K1312 were used in Experiment 1 and Experiment 2 respectively at 0.5 and 1 µg/mL as the positive control in cultures with S9. All positive controls were dissolved in Dimethyl sulphoxide (DMSO) and dosed at 1%.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle:The test material formed a solution with the solvent suitable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethyl benzanthracene (DMBA)
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
Preliminary Cytotoxicity Test: A preliminary cytotoxicity test was performed on cell cultures plated out at 3E6 cells/75 cm2 flask approximately 22 hours before dosing. On dosing, the growth media was removed and replaced with serum free media (Ham's F12). One flask per dose level was treated with and without S9 metabolic activation, 9 dose levels using halving dilutions and vehicle controls were dosed. The dose range of test material used was 19.5 to 5000 µg/ml. Exposure was for 4 hours at 37°C, after which the cultures were washed twice with phosphate buffered saline (PBS) before being trypsinised. Cells from each flask were suspended in growth medium, a sample was removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of growth medium and incubated for 7 days at 37°C ± 2°C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies. Results from the preliminary cytotoxicity test were used to select the test material dose levels for the mutagenicity experiments.

Mutagenicity Test: Several days before starting each experiment, a fresh stock of cells was removed from a liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 3E6 per 75 cm2 flask and allowed to attach overnight before being exposed to the test or control materials. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with a minimum of five dose levels of test material, and vehicle and positive controls. Treatment was for 4 hours in serum free media (Ham's F12) at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The dose range of test material was 10 to 80 µg/mL in the absence of S9 in Experiment 1 and 5 to 70 µg/mL in Experiment 2. In the presence of S9 the dose range was 10 to 100 µg/mL for both experiments. The 4-hour exposure in the presence of S9 in Experiment 2 was initially performed using a dose range of 40 to 100 µg/mL but due to unexpectedly high increased toxicity resulting from the reduction in S9 concentration insufficient dose levels survived to Day 7 for plating. To compensate for the shift in toxicity the repeat experiment used a revised and expanded dose range. At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in growth medium. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2E6 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in growth media and incubated at 37°C ± 2°C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity. During the 7 Day expression period the cultures were subcultured and maintained at 2E6 cells/225 cm2 flask on days 2 to 4 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of growth medium to determine cloning efficiency. Flasks were incubated for 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2E5 cells/75 cm2 flask (5 replicates per group) in Ham's F12 growth media (5% serum), supplemented with 10 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.
The percentages of viability and mutation frequency per survivor were calculated for each dose group. Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An assay was considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays were usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays were repeated independently, as needed, to satisfy the acceptance criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25E-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 E-6 will not be used for the evaluation of a test material.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test material clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test material must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test materials with little or no mutagenic activity, should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/mL) or twice the solubility limit of the test article in culture medium. Where a test material is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test materials that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test material, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test material.


Evaluation criteria:
Please see "ASSAY ACCEPTANCE CRITERIA" in the method section.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Preliminary cytotoxicity test: Reduction in CE of 45% at 39 µg/mL without S9 and 90% reduction with S9 at 78.1 µg/mL. Test material was toxic to cells at ≥78.1 µg/mL without S9 and at ≥156.25 µg/mL S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test: A dose range of 19.53 to 5000 µg/mL was used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in the attached Table 1. There was a dose-related reduction in the cloning efficiency (CE) in both the absence and presence of S9 with a 45% reduction at 39 µg/mL in the absence of S9 and a 90% reduction in the presence of S9 at 78.1 µg/mL. The test material was toxic to the cells at and above 78.1 µg/mL in the exposure group without S9 and at and above 156.25 µg/mL in the presence of S9.

Mutagenicity Test - Experiment 1: The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation exposure groups are presented in the attached Table 2 and Table 3. The test material achieved a reduction in cloning efficiency of 90% at 60 µg/mL in the exposure group without S9. The dose levels of 70 and 80 µg/mL were completely toxic to the CHO cells. The exposure group with S9 achieved a dose related reduction in cloning efficiency with an 89% reduction at 90 µg/mL. Although the dose level of 100 µg/mL was assessed for cloning and mutant frequencies the toxicity was greater than 90% and therefore the data generated is not considered to be relevant as it is outside the toxicity limits of the test. Neither of the vehicle control mutant frequency values were outside the acceptable range. Both the positive controls produced marked increases in the mutant frequencies per viable cell indicating the test system was operating satisfactorily and that the metabolic activation system was functional (See attached Tables 2 and 3). The test material did not induce any significant increases in the mutant frequency per viable cell which exceeded the vehicle control value by 20E-6 in either the absence or presence of metabolic activation (attached Tables 2 and 3).

Mutagenicity Test - Experiment 2: The Day 0 and Day 7 cloning efficiencies for the without and with metabolic activation exposure groups are presented in attached Tables 4 and 5. As in Experiment 1, there was dose related toxicity with the test material when compared to the vehicle controls. There was a reduction in the cloning efficiency of 78% in the absence of S9 exposure group at 45 µg/mL. The dose levels of 50, 55, 60 and 70 µg/mL achieved greater than 90% toxicity and therefore these dose levels were not assessed for cloning and mutant frequencies. In the presence of S9, a reduction in the cloning efficiency to 15% of the negative control at 70 µg/mL was achieved and this was considered to meet the study acceptance criteria for the maximum dose. The dose levels of 80, 90 and 100 µg/mL were excluded from plating on the basis of toxicity. The mutation frequency counts and mean mutation frequency per survivor per 106 cells values are presented in the attached Tables 4 and 5. In the absence and presence of metabolic activation there were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20E-6. The vehicle control values were all within the maximum upper limit of 25E-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

All tables are attached.

Due to the format and quantity of tables they have been attatched.

Conclusions:
The test material did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.
Executive summary:

Introduction.

The study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The protocol used was designed to comply with the OECD Guidelines for Testing of Chemicals No. 476'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No 440/2008 and the United Kingdom Environmental Mutagen Society. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Methods.

Chinese hamster ovary (CHO) CHO-K1 cells were treated with the test material at a minimum of eight dose levels, in duplicate, together with vehicle (solvent) and positive controls in the presence and absence of an S9 metabolic activation system. The entire experiment was repeated to confirm the result of the first experiment using modified dose levels in the absence of S9 and a reduced S9 concentration for the exposure group in the presence of S9. The dose range of the test material was selected based on the results of a preliminary cytotoxicity test and was 10 to 100 µg/mL for both the exposure groups in the presence of S9. In the absence of S9 the dose range was 10 to 80 µg/mL in Experiment 1 and 5 to 70 µg/mL in Experiment 2.

Results.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system. The test material demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion. 

The test material was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP guideline study
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
TA100- hisG46 , TA1535- hisG46, TA98- hisD3052, TA1537-hisC3076, Escherichia coli WP2uvr A- trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix: Rat liver, induced with phenobarbital and 5.6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix; 0, 0.781, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 µg/plate (TA1535 (Test 1));
0, 1.56 - 50.0 µg/plate (TA1535 (Test 2));
0, 3.13 - 100 µg/plate (TA100, TA98, TA1537);
0, 6.25 - 200 µg/plate (WP2 uvrA) +S9 mix;
0, 6.25 -200 µg/plate (TA100, TA1535, TA98, TA1537)
Vehicle / solvent:
No data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
-S9 mix; 0, 0.781, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 µg/plate (TA1535 (Test 1)); 0, 1.56 - 50.0 µg/plate (TA1535 (Test 2)); 0, 3.13 - 100 µg/plate (TA100, TA98, TA1537); 0, 6.25 - 200 µg/plate (WP2 uvrA) +S9 mix; 0, 6.25 -200 µg/plate (TA100, TA1535, TA98, TA1537)

S9: Rat liver, induced with phenobarbital and 5.6-benzoflavone
Plate/test: 3
Number of replicates: 2
Evaluation criteria:
No data
Statistics:
No data
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed at 50.0 µg/plate (TA100, TA1535, TA1537) and 100 µg/plate (TA98) without S9 mix, and at 100 µg/plate (TA100, TA1535, TA98, TA1537) with S9 mix.
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed at 50.0 µg/plate (TA100, TA1535, TA1537) and 100 µg/plate (TA98) without S9 mix, and at 100 µg/plate (TA100, TA1535, TA98, TA1537) with S9 mix.
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed at 50.0 µg/plate (TA100, TA1535, TA1537) and 100 µg/plate (TA98) without S9 mix, and at 100 µg/plate (TA100, TA1535, TA98, TA1537) with S9 mix.
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed at 50.0 µg/plate (TA100, TA1535, TA1537) and 100 µg/plate (TA98) without S9 mix, and at 100 µg/plate (TA100, TA1535, TA98, TA1537) with S9 mix.
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed at 100 µg/plate without S9 mix, and at 250 µg/plate (WP2 urvA) with S9 mix.
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Toxicity was observed at 50.0 µg/plate (TA100, TA1535, TA1537) and 100 µg/plate (TA98, WP2 urvA) without an S9 mix, and at 100 µg/plate (TA100, TA1535, TA98, TA1537) and 250 µg/plate (WP2 urvA) with an S9 mix.

The authors concluded that DVB was negative in this reverse mutation test, although styrene oxide which was structurally related to DVB was reported to be positive in the bacterial reverse mutation test and in the chromosomal aberration test, and negative in the micronucleus test on mice.

Conclusions:
DVB did not induce gene mutations in S. typhimurium or E. coli strains with and without metabolic activation
Executive summary:

The study was conducted according to the Guidelines for screening mutagenicity testing of chemicals, JAPAN. DVB did not induce gene mutations in S. typhimurium or E. coli strains with and without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP guideline study
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Chinese hamster lung (CHL/IU) cells
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Metabolic activation system:
S9 : Rat liver, induced with Phenobarbital and 5.6 - benzoflavone
Test concentrations with justification for top dose:
Dose: -S9 mix. (Continuous treatment): 0, 0.015, 0.030, 0.060 mg/mL
-S9 mix. (short-term treatment): 0, 0.0075, 0.015, 0.030 mg/mL
+S9 mix. (short-term treatment): 0, 0.015, 0.030, 0.060 mg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9 mix.: Mitomycin C, +S9 mix.: Cyclophoshamide
Details on test system and experimental conditions:
Type of cell used: Chinese hamster lung (CHL/IU) cells
Solvent: DMSO
Positive control: -S9 mix.: Mitomycin C
+S9 mix.: Cyclophoshamide
Plate/Test : 2
Evaluation criteria:
No data
Statistics:
No data
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Cytogenetic effects were not seen under the conditions of this experiment. Polyploidy was significant at 0.030 mg/mL (mid concentration) on continuous treatment.

Authors concluded that DVB did not induce polyploidy since the frequency was low (1.88%) and no significance was observed with a trend test.

Conclusions:
Cytogenetic effects were not seen under the conditions of this experiment.
Executive summary:

This study was conducted according to the Guidelines for screening mutagenicity testing of chemicals, JAPAN. Cytogenetic effects were not seen under the conditions of this experiment.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 February 2010 to 26 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP-study according to OECD guideline 476
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
To assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes

- Periodically checked for Mycoplasma contamination:yes

- Periodically checked for karyotype stability: no

- Periodically "cleansed" against high spontaneous background: yes

Cell Line
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.
Cell Culture
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.
Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.



Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
PB/betaNF S9 was prepared from the livers of male Sprague Dawley CD strain rats. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 prepreparation on the fourth day.
Test concentrations with justification for top dose:
The test material was accurately weighed and dissolved in Dimethyl sulphoxide (DMSO) and appropriate dilutions made. The test material was considered to be a complex mixture and therefore the maximum dose level was 5000 µg/mL, the maximum recommended dose level.
Vehicle and positive controls were used in parallel with the test material. Solvent treatment groups were used as the negative controls and Ethyl methane sulphonate (EMS) Sigma Batch Number 142314732109252 at 500 and 750 µg/mL was used as the positive control in cultures without S9. Dimethyl benzanthracene (DMBA) Sigma Batch Numbers 024K0757 and 105K1312 were used in Experiment 1 and Experiment 2 respectively at 0.5 and 1 µg/mL as the positive control in cultures with S9. All positive controls were dissolved in Dimethyl sulphoxide (DMSO) and dosed at 1%.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle:The test material formed a solution with the solvent suitable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Dimethyl benzanthracene (DMBA)
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
Preliminary Cytotoxicity Test
A preliminary cytotoxicity test was performed on cell cultures plated out at 3E6 cells/75 cm2 flask approximately 22 hours before dosing. On dosing, the growth media was removed and replaced with serum free media (Ham's F12). One flask per dose level was treated with and without S9 metabolic activation, 9 dose levels using halving dilutions and vehicle controls were dosed. The dose range of test material used was 19.5 to 5000 µg/mL. Exposure was for 4 hours at 37°C, after which the cultures were washed twice with phosphate buffered saline (PBS) before being trypsinised. Cells from each flask were suspended in growth medium, a sample was removed from each dose group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of growth medium and incubated for 7 days at 37°C ± 2°C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies. Results from the preliminary cytotoxicity test were used to select the test material dose levels for the mutagenicity experiments.

Mutagenicity Test
Several days before starting each experiment, a fresh stock of cells was removed from a liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 3E6 per 75 cm2 flask and allowed to attach overnight before being exposed to the test or control materials. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with a minimum of five dose levels of test material, and vehicle and positive controls. Treatment was for 4 hours in serum free media (Ham's F12) at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air. The dose range of test material was 10 to 80 µg/mL in the absence of S9 in Experiment 1 and 5 to 70 µg/mL in Experiment 2. In the presence of S9 the dose range was 10 to 100 µg/mL for both experiments. The 4-hour exposure in the presence of S9 in Experiment 2 was initially performed using a dose range of 40 to 100 µg/mL but due to unexpectedly high increased toxicity resulting from the reduction in S9 concentration insufficient dose levels survived to Day 7 for plating. To compensate for the shift in toxicity the repeat experiment used a revised and expanded dose range. At the end of the treatment period the flasks were washed twice with PBS, trypsinised and the cells suspended in growth medium. A sample of each dose group cell suspension was counted using a Coulter counter. Cultures were plated out at 2E6 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask for an estimate of cytotoxicity. Cells were grown in growth media and incubated at 37°C ± 2°C in an incubator with a humidified atmosphere of 5% CO2 in air. Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity. During the 7 Day expression period the cultures were subcultured and maintained at 2E6 cells/225 cm2 flask on days 2 to 4 to maintain logarithmic growth. At the end of the expression period the cell monolayers were trypsinised, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of growth medium to determine cloning efficiency. Flasks were incubated for 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 E5 cells/75 cm2 flask (5 replicates per group) in Ham's F12 growth media (5% serum), supplemented with 10 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The flasks were incubated for 14 days at 37°C in an incubator with a humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each flask.
The percentages of viability and mutation frequency per survivor were calculated for each dose group. Fixation and staining of all flasks was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

ASSAY ACCEPTANCE CRITERIA
An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays were usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays were repeated independently, as needed, to satisfy the acceptance criteria.
i) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but will be dependent on the scientific judgement of the Study Director. All assays below 50% cloning efficiency will be unacceptable.
ii) The background (spontaneous) mutant frequency of the vehicle controls are generally in the range of 0 to 25E-6. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35E-6 will not be used for the evaluation of a test material.
iii) Assays will only be acceptable without positive control data (loss due to contamination or technical error) if the test material clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test material must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.
iv) Test materials with little or no mutagenic activity, should include an acceptable assay where concentrations of the test material have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/mL) or twice the solubility limit of the test article in culture medium. Where a test material is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test materials that are clearly mutagenic.
v) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for viable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.
vi) Five dose levels of test material, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test material.


Evaluation criteria:
Please see "ASSAY ACCEPTANCE CRITERIA" in the method section.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Preliminary Cytotoxicity Test: Dose-related reduction in CE. Without S9: no toxicity at 39.06 μg/mL and 99% reduction in CE at 78.13 μg/mL. With S9: 69% reduction in CE at 78.13 μg/mL. Test material was toxic to cells at ≥156.25 μg/mL in both groups.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test: A dose range of 19.53 to 5000 μg/mL was used in the preliminary cytotoxicity test. The results of the individual flask counts and their analysis are presented in Table 1. It can be seen that there was a dose-related reduction in the cloning efficiency (CE) in both the presence and absence of S9. In the absence of S9 the toxicity curve was very steep with no toxicity at 39.06 μg/mL and a 99% reduction in cloning efficiency at 78.13 μg/mL. In the presence of S9 the toxicity was less marked with a 69% reduction in cloning efficiency at 78.13 μg/mL. The test material was toxic to the cells at and above 156.25 μg/mL in both exposure groups. A precipitate of test material was seen at the end of exposure in both exposure groups at and above 78.13 μg/mL which became greasy/oily at and above 625 μg/mL.

Mutagenicity Test - Experiment 1: The Day 0 and Day 7 cloning efficiencies are presented in Table 2 and Table 3. The test material achieved a reduction in cloning efficiency of 72% at 50 μg/mL in the exposure group without S9. The dose levels of 60, 70 and 80 μg/ml were not plated due to excessive toxicity. In the exposure group in the presence of S9 a reduction in cloning efficiency of 78% was achieved at 90 μg/mL with the higher dose level of 100 μg/mL being omitted from plating due to excessive toxicity. The dose levels of 20 and 30 μg/mL were considered to be surplus to the requirements of the test and were therefore not selected for plating. Precipitate was not seen in either exposure group at the end of the exposure period, although a cloudy precipitate was noted in the absence of S9 at and above 60 μg/ml after dosing. Vehicle control values were all within the maximum upper limit of 25E-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected. The mutation frequency counts and mean mutation frequency per survivor values are presented in Table 2 and Table 3. There were no group increases in mutation frequency per survivor which exceeded the vehicle control value by 20E-6 with or without the presence of S9.

Mutagenicity Test - Experiment 2: The Day 0 and Day 7 cloning efficiencies are presented in Tables 4 and 5. It can be seen that the toxicity in the absence of S9 was similar to that seen in Experiment 1 with an 87% reduction in cloning efficiency being achieved at 55 μg/mL. The dose levels of 60 μg/mL and 70 μg/mL achieved greater than 90% toxicity and were therefore excluded from plating. In the presence of S9 the toxicity was greater than that seen in Experiment 1, probably as a result of the reduced S9 concentration. The dose level of 75 μg/mL achieved a reduction in cloning efficiency of 84% whilst the higher dose levels of 80, 85, 90, 95 and 100 μg/mL exceeded 90% and were therefore not selected for plating for cloning and mutant frequencies. Precipitate was seen at the beginning and end of exposure at and above 50 μg/mL in the absence of S9 only. The mutation frequency counts and mean mutation frequency per survivor per 106 cells values are presented in Tables 4 and 5. In the absence and presence of metabolic activation there were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20E-6. It can be seen that the vehicle control values were all within the maximum upper limit of 25E-6 mutants per viable cell, and that the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

All tables are attached.

Due to the format and quantity of tables they have been attatched.

Conclusions:
The test material did not induce significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test material was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.
Executive summary:

Introduction: The study was conducted to assess the potential mutagenicity of the test material on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The protocol used was designed to comply with the OECD Guidelines for Testing of Chemicals No. 476 'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No 440/2008 and United Kingdom Environmental Mutagen Society. The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6-TG) as the selective agent.

Methods: Chinese hamster ovary (CHO) CHO-Kl cells were treated with the test material at a minimum of eight dose levels, in duplicate, together with vehicle (solvent) and positive controls in the presence and absence of an S9 metabolic activation system. The entire experiment was repeated to confirm the result of the first experiment using modified dose levels in the absence of S9 and a reduced S9 concentration with modified dose levels for the exposure group in the presence of S9. The dose ranges of test material were selected based on the results of a preliminary cytotoxicity test and were 5 to 80 μg/mL in the absence of S9 and 20 to 100 μg/mL in the presence of S9 in Experiment 1. In Experiment 2 the dose ranges were 10 to 70 μg/mL and 30 to 100 μg/mL in the absence and presence of metabolic activation respectively.

Results: The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system. The test material demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion: The test material was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP-study equivalent to OECD guideline 471.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay
Target gene:
TA97 (hisD6610) and his01242), TA98 (hisD3052), TA100 (hisG46), TA1535 (hisG46), and TA1537 (hisC3076)
WP2 uvrA pKM101 (trp)
Species / strain / cell type:
other: Salmonella typhimurium tester strains TA97, TA98, TA100, TA1535, and TA1537 and with the E. coli tester strain WP2 uvrA pKM101
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver)
Test concentrations with justification for top dose:
BioReliance Corporation (Zieger et al. 1987) : 0, 0.3, 1, .3, 10, 33 or 100 µg/plate
SRI International: 0, 0.3, 1, .3, 10, 33 or 100, 333, 666, or 1000 µg/plate
SITEK Research Laboratories: 0,5,10, 25, 50, 75, 100, 250, 500, or 750 µg/plate
Vehicle / solvent:
no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: SRI International: Positive controls in absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA97 and TA1537), and 4-nitro-o-phenylenediamine (TA98). Positive control for metabolic activation all strains 2-aminoanthracene
Remarks:
SITEK Research Lab.:Positive controls in the absence of metabolic activation were sodium azide (TA100 ), 4-nitro-o-phenylenediamine (TA98), and methyl methanesulfonate (E. coli). Positive control for metabolic activation with all strains 2-aminoanthracene
Details on test system and experimental conditions:
Three independent mutagenicity assays were conducted with divinylbenzene. Testing was performed for the first two assays with divinylbenzene of unknown purity as reported by Zeiger et al. (1987). The third assay, conducted with the same lot of divinylbenzene (80%) tested in the 2-year study, used a slightly modified protocol (activation only with rat liver S9) and also employed Escherichia coli strain WP2 uvrA pKM101 as a bacterial tester strain in addition to Salmonella typhimurium strains. Divinylbenzene was sent to the laboratories as a coded aliquot from Radian Corporation (Austin, TX). It was incubated with the Salmonella typhimurium tester strains TA97, TA98, TA100, TA1535, and TA1537 and with the E. coli tester strain either in buffer or S9 mix (metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver) for 20 minutes at 37° C. Top agar supplemented with L-histidine and d-biotin was added, and the contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C.

Each trial consisted of triplicate plates of concurrent positive and negative controls and five doses of divinylbenzene. The high dose was limited by toxicity. All trials were repeated at the same or a higher S9 fraction.
Evaluation criteria:
In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose-related, is not reproducible, or is not of sufficient magnitude to support a determination of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive.
Statistics:
no data
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
None

The highest concentration tested at one laboratory was 100 μg/plate; the other two laboratories tested higher concentrations, up to 1000 μg/plate. It should be considered that inadequate exposure of the tester strains may have occurred, as incubation with this volatile compound was not carried out within the closed environment of a desiccator.

Conclusions:
Divinylbenzene-HP was not mutagenic in any of three independent gene mutation assays using Salmonella typhimurium strains TA97, TA98, TA100, TA1535, or TA1537 or Escherichia coli tester strain WP2 uvrA with or without induced hamster or rat liver enzymes.
Executive summary:

Three independent mutagenicity assays were conducted with divinylbenzene. Testing was performed for the first two assays with divinylbenzene of unknown purity as reported by Zeiger et al. (1987). The third assay, conducted with the same lot of divinylbenzene (80%) tested in the 2-year study, used a slightly modified protocol (activation only with rat liver S9) and also employed Escherichia coli strain WP2 uvrA pKM101 as a bacterial tester strain in addition to Salmonella typhimurium strains.

Divinylbenzene-HP was not mutagenic in any of three independent gene mutation assays using Salmonella typhimurium strains TA97, TA98, TA100, TA1535, or TA1537 or Escherichia coli tester strain WP2 uvrA with or without induced hamster or rat liver enzymes.

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

Genetic toxicity in vivo

Description of key information

3 in vivo cytogenicity studies conducted with DVB-55 and DVB-HP are available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10/2009-07/2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP-study according to OECD guideline 474.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, North Carolina)
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: 25.8 g (mean)
- Assigned to test groups randomly: yes
- Fasting period before study: not applicable
- Housing: animals were housed one per cage in stainless steel cages. Cages had wire mesh floors and were suspended above absorbent paper. Non-woven gauze was placed in the cages to provide a cushion from the flooring for rodent feet. The gauze provided environmental enrichment. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Route of administration:
inhalation: vapour
Vehicle:
not applicable
Details on exposure:
Chambers: the animals were exposed to filtered air or DVB-55 vapors in 4 cubic meter stainless steel and glass Rochester-type whole-body exposure chambers [1.5 meters (m) x 1.5 m wide x 1.3 m deep with a pyramidal top and bottom]. Chamber airflow was maintained at approximately 900 liters per minute. This flow rate was sufficient to provide the normal concentration of oxygen to the animals and 12-15 calculated air changes per hour. The chambers were operated at a slightly negative pressure, relative to the surrounding area. The airflow through the exposure chamber was calibrated with a gas meter (Singer Aluminum Diaphragm Meter, Model AL-1400, American Meter Division, Philadelphia, Pennsylvania) prior to the start of the study. Chamber temperature and relative humidity data were recorded from a thermometer (Control Company, Friendswood, Texas) and hygrometer (Brooklyn Thermometer Co., Inc., Farmingdale, New York) stationed in the interior of each chamber. The chamber temperature and relative humidity was controlled by a system designed to maintain values of approximately 22 ± 3 °C and 30% – 70%, respectively. Chamber temperature, relative humidity, and airflow data were recorded once per hour during each exposure period.
Generation System: The various concentrations of DVB-55 were generated using a glass J-tube method. Liquid test material was pumped into the glass J-tube assembly and vaporized by the flow of compressed nitrogen gas passing through the bead bed of the glass J-tube. The nitrogen was heated with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. All chambers, including the 0 ppm (negative control) chamber received the same volume percent of supplemental nitrogen (carrier gas). The minimum amount of nitrogen necessary to reach the desired chamber concentrations were used. The generation system was electrically grounded and the J-tubes were changed as needed. The vaporized test material and carrier gas was mixed and diluted with supply air to achieve a total flow of 900 liters per minute at the desired test chamber concentration.
Duration of treatment / exposure:
3 consecutive days
Frequency of treatment:
6 hours/day
Post exposure period:
2 and 48 hours after the termination of the third exposure
Dose / conc.:
37.5 ppm (nominal)
Remarks:
35.0 ± 3.0 ppm (analytical)
Dose / conc.:
75 ppm (nominal)
Remarks:
74.1 ± 4.3 ppm (analytical)
Dose / conc.:
150 ppm (nominal)
Remarks:
142.9 ± 7.9 ppm (analytical)
No. of animals per sex per dose:
5/exposure/timepoint, except for highest exposure where 6/timepoint were used
Control animals:
yes, sham-exposed
Positive control(s):
Cyclophosphamide monohydrate (CP), CAS Number 6055-19-2, Source: Sigma, St. Louis, Missouri.

CP was administered by oral gavage in an aliquot of 10 mL/kg body weight (bw). CP when administered by oral gavage has been shown to induce micronuclei in polychromatic erythrocytes. The dose level for CP was 40 mg/kg and was based upon experience which indicated a pronounced micronuclei induction in B6C3F1/Crl mice at this dose level. CP was administered only once, approximately 48 hours (±1 hour) before sacrifice. A frozen stock solution of CP dissolved in distilled water (thawed and brought to room temperature prior to use) was used.
Tissues and cell types examined:
Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry with traditional blood smears prepared as a backup. Approximately 5000 RETs were analyzed per blood sample. The number of normochromatic erythrocytes (NCE), MN-NCE, RET, and MN-RET were recorded for each sample and the frequency of MN-RET was determined to provide an indication of genotoxic potential. The frequency of RETs relative to total erythrocytes was determined to provide an indication of perturbations in hematopoetic activity indicative of cell toxicity. For each of the treatment groups, a mean and standard deviation was calculated to describe the frequency of RET and MN-RET observed. The analyses were conducted utilizing a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter).
Details of tissue and slide preparation:
Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry with traditional blood smears prepared as a backup. Samples were prepared and analyzed per instructions in the mouse MicroFlow Micronucleus Analysis Kit Manual (Litron Laboratories, Rochester, New York). At the end of the specified interval following treatment, a peripheral blood sample was collected from the orbital sinus of all surviving animals into anticoagulant solution following anesthesia with isoflurane. The blood samples were fixed in ultracold (-70 to -80°C) methanol within five hours of collection. All fixed blood samples were stored at -80°C. Fixed blood samples were washed with a cold, buffered salt solution and isolated by centrifugation. The resulting cell pellets were stored at 4°C until staining. Blood samples were ultimately incubated with RNAse to degrade the high levels of RNA present in the reticulocytes (RET) and a fluorescently labeled antibody to the transferrin receptor (anti-CD71-FITC) to specifically identify the RET. A propidium iodide solution was added to each sample immediately before flow cytometry (FCM) analysis to stain the DNA, including that of micronuclei. Blood samples were analyzed by high-speed FCM. In this system, the sample was moved at a high velocity past a laser set to provide 488 nm excitation. The fluorescent wavelengths emitted by each cell were collected by photomultiplier tubes. Using the previously described staining procedure, the propidium iodide-stained DNA of the micronuclei emitted a red fluorescence and the anti-CD71-FITC antibody emitted a high green fluorescent signal permitting differentiation between cells with and without micronuclei. In addition to obtaining fluorescent profiles, FCM simultaneously provided cell size information by determining the light scatter properties of each cell or combination of cells.
Evaluation criteria:
A test was considered valid if all of the following conditions were met: the range of MN-RET values in the negative controls were within reasonable limits of the recent laboratory background range. There was a significant increase in the incidence of MN-RET in the positive control treatment as compared to the concurrent negative controls. The mean for percent RET value in one or more of the test material treated groups was greater than or equal to 20% of the control value indicating no undue effect on erythropoiesis (toxicity). A test material was considered positive in this assay if the following criterion was met: statistically significant increase in MN-RET frequency at one or more dose levels accompanied by a dose response. A test material was considered negative in this assay if the following criterion was met: no statistically significant dose-related increase in MN-RET when compared to the negative control. A test result not meeting the criteria for either the positive or the negative response was considered to be equivocal.
Statistics:
Descriptive statistics only (means and standard deviations) were reported for chamber concentration, temperature, humidity, airflow, and body weights. MN-RET and percent RET were tested for equality of variance using Bartlett's test (alpha = 0.01). If the results from Bartlett's test were significant, then the data for the parameter may have been subjected to a transformation to obtain equality of the variances. The transformations that were examined are the common log, the inverse, and the square root in that order. The data was reviewed and an appropriate form of the data selected and subjected to the following analysis. The MN-RET data and the data on percent RET were analyzed by a one-way analysis of variance. Pairwise comparisons of treated vs. control groups were done, if the dose effect was significant, by Dunnett’s t-test, one-sided (upper) for MN-RET and two-sided for the percent RET. Linear dose-related trend tests were performed if any of the pairwise comparisons yield significant differences. The alpha level at which all tests were conducted was 0.05. The MN-NCE was not analyzed statistically and was only used as an adjunct end point to evaluate the biological significance of the MN-RET results. The final interpretation of biological significance of the responses is based on both statistical outcome and scientific judgment.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Mean chamber concentration values for the 0, 37.5, 75 and 150 ppm chambers were 0, 35.0 ± 3.0, 74.1 ± 4.3, or 142.9 ± 7.9 ppm DVB-55 (study mean), respectively. The actual exposure concentrations ranged from approximately 93 to 98% of the targeted values. Animals that were sacrificed two hours after the final exposure had body weight decreases of more than 10% in all exposure levels. Body weights of animals sacrificed 48 hours after the final exposure were minimally affected. On the initial day of exposure animals in the 37.5 and 75 ppm exposure groups exhibited decreased activity. These same mice had decreased faeces on the second day of exposure and no remarkable clinical observations by the third day. In the highest exposure group (150 ppm), 7 out of 12 mice displayed decreased activity on the initial day of exposure. By the second day, all mice at the highest exposure level had a decrease in the quantity of faeces, however, these mice had no remarkable clinical observations by the third day. Three hours after the initial exposure decreases in body temperature were observed in all test material exposure groups (up to 9.2°C). The body temperature of the animals was minimally affected following subsequent exposures after this initial body temperature decrease. There were no significant differences in MN-RET frequencies between the groups exposed with the test material and the negative controls. The adequacy of the experimental conditions for the detection of induced micronuclei was ascertained from the observation of a significant increase in the frequencies of micronucleated RETs in the positive control group. The percent RET values observed in the test material-exposed animals were significantly decreased from the negative control values in all exposure groups. The percent RET values of the positive control animals were found to be significantly lower than those of the negative control animals. Treatment-related toxicity was observed in male mice administered three consecutive targeted daily exposures of DVB-55 up to 150 ppm (actual exposure was142.9 ± 7.9 ppm). All exposure groups had a significant decrease in the percent RET indicating that DVB-55 was systemically available to the target tissue. Based upon the results of the study reported herein, it was concluded that the test material, DVB-55, did not induce a significant increase in the frequencies of micronucleated reticulocytes in the peripheral blood when given as a six hour inhalation exposure on three consecutive days to male B6C3F1/Crl mice. Hence, DVB-55 is considered negative in this test system under the experimental conditions used.
Conclusions:
DVB-55, did not induce a significant increase in the frequencies of micronucleated reticulocytes in the peripheral blood when given as a six hour inhalation exposure on three consecutive days to male B6C3F1/Crl mice. Hence, DVB-55 is considered negative in this test system under the experimental conditions used.
Executive summary:

The in vivo genotoxic potential of DVB-55 (a mixture of diethenylbenzene and ethenylethylbenzene) was evaluated by examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood. In a previously published study, Kligerman et al. (1996, Mutat. Res, 370:107-113) showed a weak positive effect for the induction of micronuclei following inhalation exposure of mice to DVB-55. In the current study, male B6C3F1/Crl mice were whole-body exposed to actual concentrations of 0, 35.0 ± 3.0, 74.1 ± 4.3, or 142.9 ± 7.9 ppm DVB-55 six hours/day for three days. The exposure concentrations were selected to overlap those employed by Kligerman et al. with the highest concentration representing the maximum tolerated exposure concentration. Peripheral blood from exposed mice was sampled 2 and 48 hours after the final exposure. The first sampling time was selected to replicate the timing of the Kligerman et al. study and the second time was consistent with the OECD test guidelines. All animals were observed for clinical signs prior to the exposure, immediately following the exposure, approximately three hours following each exposure, and daily after the final exposure. Groups of mice, five/exposure/timepoint, except the highest exposure where six/timepoint, were sacrificed approximately 2 and 48 hours after the final exposure for the collection of peripheral blood and evaluation of RET (approximately 5000/animal) for MN by flow cytometry. The proportion of RET was determined based upon 5000 RET per animal and the results expressed as a percentage. Mice treated with 40 mg/kg bw cyclophosphamide monohydrate by a single oral gavage dose and sacrificed 48 hours later served as positive controls. All animals survived to the end of the observation period. Treatment-related clinical signs including decreased activity, decreased faeces, and decreased body temperatures were observed in all test material exposure groups. There were no statistically significant increases in the frequencies of MN-RET in groups exposed to the test material and sampled at approximately 2 and 48 hours after the final exposure. There were statistically significant decreases in the percent RET in all test material exposure groups. There was a significant increase in the frequency of MN-RET and a decrease in the percentage of RET in the positive control chemical group as compared to the negative control group. Under the experimental conditions used, DVB-55 was considered to be negative in the mouse peripheral blood micronucleus test via inhalation exposure.

Endpoint:
genetic toxicity in vivo, other
Remarks:
chromosome aberration and DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The effect of the test substance in vivo on sister chromatid exchange induction, micronuclei frequencies, and indices of cellular toxicity in mice hematopoietic cells was studied.
GLP compliance:
not specified
Type of assay:
other: micronucleus assay and sister chromatid exchange
Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals or test system and environmental conditions:
B6C3F1 mice (Charles River, Portage, MI) were exposed to DVB-55 or conditioned air for 6 h per day for 3 days in Hazleton 1000 inhalation chambers.
Food (NIH-07) was removed during chemical exposure; chlorinated, filtered, UV-disinfected tap water was provided ad libitum.
Route of administration:
inhalation: aerosol
Vehicle:
none
Details on exposure:
Liquid DVB-55 was vaporized in the exposure chamber (Hazleton 1000 inhalation chambers) air supply, the vapour was mixed with conditioned air (HEPA-filtered, charcoal-scrubbed, temperature- and humidity-controlled), and delivered to the exposure chambers.
Duration of treatment / exposure:
3 days
Frequency of treatment:
6 hours/day
Post exposure period:
None
Dose / conc.:
25 ppm (nominal)
Remarks:
30.7 ± 5.6 (analytical)
Dose / conc.:
50 ppm (nominal)
Remarks:
53.0 ± 2.1 ppm (analytical)
Dose / conc.:
75 ppm (nominal)
Remarks:
78.2 ± 3.3 ppm (analytical)
No. of animals per sex per dose:
5 males/dose
Control animals:
yes
Positive control(s):
None
Tissues and cell types examined:
blood (tail) smears, polychromatic erythrocytes-micronucleus (MN) analysis
spleens, splenocytes- sister chromatid exchange (SCE) and chromosome aberration (CA) analyses
Details of tissue and slide preparation:
Within 2 hours following the last exposure, the mice were killed by carbon dioxide asphyxiation. The tip of the tail of each animal was severed, and blood smears were made with one drop of blood mixed with 1 drop of fetal bovine serum (HI-FBS; GIBCO, Grand Island, NY). Slides were air dried, stained in 0.01 mg/mL acridine orange in phosphate-buffered saline (pH 7.4) for 5 min, and rinsed with cold tap water. The slides were wet-mounted with phosphate-buffered saline (pH 7.4) and analyzed using fluorescence microscopy. Immediately following blood smear preparation, the spleen was removed from each animal and minced in 2.0 mL RPM1 1640 (GIBCO). Cell suspensions were counted, and 3 million cells per animal were cultured in 2.0 mL RPM1 1640 with L-glutamine with 25 mM HEPES fortified with 15% HI-FBS, 1% penicillin-streptomycin (GIBCO), an additional 2 mM L-glutamine (GIBCO), and 4 µg/mL concanavalin A (Type IV, Sigma Chemical Company, St. Louis, MO). Cultures were incubated at 37°C with 5.0% CO2. At 21 hours post-initiation, 20 µL of a 1 mM 5-bromo-2’-deoxyuridine solution were added to each culture. Cells were harvested at 48 hours post-initiation by centrifugation, treated with hypotonic KCl, and fixed using standard procedures. Slides were stained for chromatid differentiation by the fluorescence-plus-Giemsa procedure, and coded so that the scorers did not know the identification of the slides scored. For SCE, CA, and replicative index analyses, 25 second-division metaphase, 100 first-division metaphases, and 100 metaphases were analyzed, respectively. One thousand PCEs were scored for the presence or absence of MN, and 500 erythrocytes were observed to determine the percentage of PCEs.
Evaluation criteria:
No data
Statistics:
For all treatments the animal was considered the unit of experimentation. Linear regression analysis was used to determine if there was a dose-related increase in any of the cytogenetic endpoints. If the regression analyses was significant, a one-way analysis of variance was performed, and the least significant difference test was applied to compare treatments to the concurrent control. All these tests were one-tailed and the alpha level was set at 0.05. To determine if the percentage of PCEs or the replicative index was affected by the dosing, a one-way analyses of variance was performed, the alpha level set at 0.05, and the tests were two-tailed. All statistical analyses were conducted on a personal computer using Statgraphics Plus@ software (Manugistics, Rockville, MD).
Sex:
male
Genotoxicity:
other: weak
Toxicity:
no effects
Remarks:
There was no indication of toxicity as measured by cell cycle kinetics in the splenocytes or the percentage of polychromatic erythrocytes in the peripheral blood smears.
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Effect of DVB-55 inhalation on sister chromatid exchange induction, micronuclei frequencies, and indices of cellular toxicity in mice hematopoietic cells (Mean ± SD)

Conc. DVB SCEs/metaphase Replicate index MN/1000 PCEs %PCEs
Control 11.6 ± 1.3 1.22 ± 0.08 4.0 ± 2.1 2.5 ± 0.9
30.7 ppm 12.0 ± 2.3 1.19 ± 0.05 10.6 ± 0.34* 2.0 ± 0.8
53.0 ppm 13.8 ± 1.1* 1.15 ± 0.02 13.6 ± 4.4 * 2.1 ± 0.2
78.2 ppm 14.5 ± 1.4* 1.14 ± 0.10 9.0 ± 1.4 2.0 ± 0.8

* Statistically different from control (p < 0.05) by the least significant difference test.

Effect of DVB-55 inhalation on chromosome aberrations in splenocytes of mice

Conc. DVB % Aberrant Chromatid deletions(per 100) Chromatid exchanges (per 100) Chromosome deletions (per 100)
Control 1.6 ± 0.6 0-1 per animal 0-1 per animal 0-1 per animal
30.7 ppm 3.2 ± 1.6 1-3 per animal 0-1 per animal 0-1 per animal
53.0 ppm 5.2 ± 3.8* 0-8 per animal 0-1 per animal 0-1 per animal
78.2 ppm 4.4 ± 1.5* 3-5 per animal 0-1 per animal 0-1 per animal

* Statistically different from control ( p < 0.05) by the least significant difference test.

Data from the in vivo exposure of mice to DVB-55 indicate that DVB-55 is a weak genotoxicant. The SCE analysis shows that DVB-55 caused a linear dose-related increase in SCE frequency with 53 ppm being the lowest concentration that gave a statistically significant increase over the concurrent controls. The linear correlation coefficient was 0.61. DVB-55 appears to be mildly clastogenic. The two highest concentrations were significantly different from the controls, but the responses did not increase in a stepwise fashion. The increase in CAs was manifest as an increase in chromatid deletions only. There was no evidence of an increase in either chromosome-type aberrations or chromatid exchanges. Similarly, there was a statistically- significant increase in the frequency of micronucleated PCEs. Again there was not a monotonic increase in response with dose; the low and middle doses were statistically different from the control, and the highest dose just failed to reach statistical significance. Both measures of cytotoxicity - the replicative index and the percentage of PCEs - gave no evidence of a slowing of the cell cycle or inhibition of bone marrow mitogenesis.

Conclusions:
DVB-55 induced a dose-dependent increase in SCE with the two highest doses reaching statistical significance. Similarly, there was a statistically significant although less pronounced increase in the frequency of CAs in splenocytes and MN in polychromatic erythrocytes. There was no indication of toxicity as measured by cell cycle kinetics in the splenocytes or the percentage of polychromatic erythrocytes in the peripheral blood smears. Thus, DVB-55 appears to be a weak genotoxicant in vivo.
Executive summary:

B6C3F1 mice were exposed to DVB-55 (25, 50, or 75 ppm) or conditioned air for 6 h per day for 3 days in Hazleton 1000 inhalation chambers. Liquid DVB-55 was vaporized in the exposure chamber (Hazleton 1000 inhalation chambers) air supply, the vapour was mixed with conditioned air (HEPA-filtered, charcoal-scrubbed, temperature- and humidity-controlled), and delivered to the exposure chambers. Data from the in vivo exposure of mice to DVB-55 indicate that DVB-55 is a weak genotoxicant. Polychromatic erythrocytes were examined from micronucleus (MN) analysis and splenocytes were examined for sister chromatid exchange (SCE) and chromosome aberrations (CA). The SCE analysis shows that DVB-55 caused a linear dose-related increase in SCE frequency with 53 ppm being the lowest concentration that gave a statistically significant increase over the concurrent controls. The linear correlation coefficient was 0.61. DVB-55 appears to be mildly clastogenic. The two highest concentrations were significantly different from the controls, but the responses did not increase in a stepwise fashion. The increase in CAs was manifest as an increase in chromatid deletions only. There was no evidence of an increase in either chromosome-type aberrations or chromatid exchanges. Similarly, there was a statistically- significant increase in the frequency of micronucleated PCEs. Again, there was not a monotonic increase in response with dose; the low and middle doses were statistically different from the control, and the highest dose just failed to reach statistical significance. Both measures of cytotoxicity - the replicative index and the percentage of PCEs - gave no evidence of a slowing of the cell cycle or inhibition of bone marrow mitogenesis.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP-study equivalent to OECD guideline 474.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
A detailed discussion of this assay is presented by MacGregor et al. (1990). Peripheral blood samples were obtained from male and female mice from the 3-month toxicity study previously described in section 7.5.3 Report NTP TR 534
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female B6C3F1 mice were obtained from Taconic (Germantown, NY). On receipt, the mice were 4 weeks old. Animals were quarantined for 13 or 14 days and were 6 weeks old on the first day of the studies. Feed was available ad libitum except during exposure periods; water was available ad libitum. All animals were housed individually.
Route of administration:
inhalation: vapour
Vehicle:
None
Details on exposure:
Mice were exposed to divinylbenzene-HP at concentrations of 0, 12.5, 25, 50, 100, or 200 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks.

Preheated divinylbenzene-HP was pumped onto glass beads in a heated glass column where it was vaporized. Heated air flowed through the column and carried the vapour out of the generator. Generator output was controlled by the delivery rate of the chemical metering pump. Buildup and decay rates for chamber vapour concentrations were determined with animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapour generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapour generation was terminated (T10) was approximately 12.5 minutes. Based on experimental data, a T90 value of 12 minutes was selected for all studies. Throughout the studies, concentration uniformity, persistence and stability of the chemical, and degradation impurities were monitored in the chambers. Chamber concentration uniformity was maintained; no degradation was observed, and no impurities other than those in the bulk chemical were observed.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours plus T90 (12 minutes) per day, 5 days per week
Post exposure period:
None
Dose / conc.:
12.5 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes
Positive control(s):
No data
Tissues and cell types examined:
peripheral blood samples
Details of tissue and slide preparation:
At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of 10 animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined as a measure of bone marrow toxicity.
Evaluation criteria:
In the micronucleus test, an individual trial was considered positive if the trend test P value was less than or equal to 0.025 or if the P value for any single exposed group was less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction wass preferably based on reproducibly positive trials. Results of the 3-month study were accepted without repeat tests, because additional test data could not be obtained. Ultimately, the final call was determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.
Statistics:
The results were tabulated as the mean of the pooled results from all animals within an exposure group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
No increases in the frequencies of micronucleated NCEs or alterations in the percentages of PCEs were seen in peripheral blood of male or female B6C3F1 mice exposed to divinylbenzene by inhalation for 3 months
Conclusions:
No increases in the frequencies of micronucleated NCEs or alterations in the percentages of PCEs were seen in peripheral blood of male or female B6C3F1 mice exposed to divinylbenzene by inhalation for 3 months
Executive summary:

A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of 10 animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined as a measure of bone marrow toxicity.

No increases in the frequencies of micronucleated normochromatic erythrocytes or alterations in the percentages of polychromatic erythrocytes were seen in peripheral blood of male or female B6C3F1 mice exposed to divinylbenzene-HP by inhalation for

3 months.

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

Additional information

in vitro: Several good quality in vitro gene mutation studies (Klimisch 1 and 2) in bacteria, yeast and mammalian cells are available for different grades of the reaction mass of divinylbenzene and ethylstyrene. All studies reported negative results for gene mutation. One in vitro chromosomal aberration study (Klimisch 1) is available for DVB-96 reporting negative results.

In vivo: The in vivo genotoxic potential of DVB-55 (a mixture of diethenylbenzene and ethenylethylbenzene) was evaluated by examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood. In a previously published study, Kligerman et al. (1996, Mutation Research, 370, 107-113) showed a weak positive effect for the induction of micronuclei following inhalation exposure of mice to DVB-55. In the current study, male B6C3F1/Crl mice were whole-body exposed to actual concentrations of 0, 35.0 ± 3.0, 74.1 ± 4.3, or 142.9 ± 7.9 ppm DVB-55 six hours/day for three days. The exposure concentrations were selected to overlap those employed by Kligerman et al. with the highest concentration representing the maximum tolerated exposure concentration. Peripheral blood from exposed mice was sampled 2 and 48 hours after the final exposure. The first sampling time was selected to replicate the timing of the Kligerman et al. study and the second time was consistent with the OECD test guidelines. All animals were observed for clinical signs prior to the exposure, immediately following the exposure, approximately three hours following each exposure, and daily after the final exposure. Groups of mice, five/exposure/timepoint, except the highest exposure where six/timepoint, were sacrificed approximately 2 and 48 hours after the final exposure for the collection of peripheral blood and evaluation of RET (approximately 5000/animal) for MN by flow cytometry. The proportion of RET was determined based upon 5000 RET per animal and the results expressed as a percentage. Mice treated with 40 mg/kg bw cyclophosphamide monohydrate by a single oral gavage dose and sacrificed 48 hours later served as positive controls. All animals survived to the end of the observation period. Treatment-related clinical signs including decreased activity, decreased faeces, and decreased body temperatures were observed in all test material exposure groups. There were no statistically significant increases in the frequencies of MN-RET in groups exposed to the test material and sampled at approximately 2 and 48 hours after the final exposure. There were statistically significant decreases in the percent RET in all test material exposure groups. There was a significant increase in the frequency of MN-RET and a decrease in the percentage of RET in the positive control chemical group as compared to the negative control group. Under the experimental conditions used, DVB-55 was considered to be negative in the mouse peripheral blood micronucleus test via inhalation exposure.

In the more recent in vivo micronucleus study with DVB-55 (Dow 2010) a decrease in body temperature 3 hours after the initial exposure was observed in the high dose groups. Hypothermia may lead to an increase in micronuclei as an indirect result of this physiological change and it has been hypothesized that clastogenic injury may be caused by interference with microtubule assembly and spindle function (Asanami S and Shimono K (1997). Mutation Research, 390:70-83 and Asanami S, Shimono K, and Kaneda S (1998). Mutation Research, 413:7-14). Although this effect has not been observed in this recent study it may have caused the slight increase in micronuclei reported by Kligerman et al (1996). No body temperature data has been reported by Kligerman. Hence, it cannot be excluded that the slight positive effects reported by Kligerman were artefacts due to hypothermia.

This is also supported by the results of an in vivo micronucleus test conducted with DVB-80 as part of an NPT bioassay (2006) where no increase in the frequency of micronuclei was seen in peripheral blood of male or female B6C3F1 mice exposed to up to 200 ppm DVB-80 by inhalation for 3 months.

Justification for classification or non-classification

No genotoxicity of DVB/EVB was reported in any of the in vitro gene mutation studies conducted with several grades of the reaction mass of divinylbenzene and ethylstyrene. In a weight of evidence evaluation of the 3 in vivo cytogenicity studies conducted with DVB-55 and DVB-HP it has been concluded that the different grades of the reaction mass do not have the potential to induce clastogenic effects in vivo. Therefore, no classification is required for any grade of the test substance according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/208.