Divinylbenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

The test chemical did not induce gene mutation in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA - in the presence and absence of rat liver activation system and hence the chemical is not likely to classify as a gene mutant in vitro and hence is not likely to classify as a gene mutant in vitro.

Chromosome aberration study:

the test chemical did not induce chromosome aberrations in the Chinese hamster lung (CHL/IU) cells in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro and hence is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from J-check

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Guidelines for Screening Toxicity Testings of Chemicals (Japan) and OECD Guidelines No. 471 and 472

Principles of method if other than guideline:

Gene mutation test was conducted on Salmonella typhimurium TA 100, TA 1535, TA 98, TA 15371 and Escherichia coli WP 2 uvr A 2 by using test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

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, TA1 535, TA98, TA1537); 0, 15.6 - 500 μg/plate(WP2 uvrA)

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide and 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; preincubationDURATION- Preincubation period: 20 minutes- Exposure duration: 48 hoursNUMBER OF REPLICATIONS: 2 METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 0.1 mL of the test substance preparation solution, 0.5 mL of the phosphate buffer solution (0.5 mL of the S 9 mix in the S9 mix addition test) and 0.1 mL of the test bacterium solution were mixed in a small test tube and preincubated at 37 ° C. for 20 minutes , Add 2 mL of top agar and mix and sonicate it on a synthetic medium flat plate.

Evaluation criteria:

Among the five test bacteria used, in the S9 mix-free test or S9 mix addition test of one or more test bacteria, the average value of the number of mutant colonies on the flat plate containing the test substance was 2 Fold or more, and when reproducibility and dose dependency were observed in the increase, the test substance was judged to be mutagenic (positive) in this test system.

Species / strain:

S. typhimurium, other: TA100, TA1535, TA98, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: Non mutagenic

Conclusions:

The test chemical did not induce gene mutation in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA - in the presence and absence of rat liver activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA with and without Rat liver, induced with phenobarbital and 5,6-benzoflavone activation system. 0.1 mL of the test substance preparation solution, 0.5 mL of the phosphate buffer solution (0.5 mL of the S 9 mix in the S9 mix addition test) and 0.1 mL of the test bacterium solution were mixed in a small test tube and preincubated at 37 ° C. for 20 minutes, Add 2 mL of top agar and mix and sonicate it on a synthetic medium flat plate. As a control group, a solvent to be used or several kinds of positive control substance solutions was used in place of the test substance preparation solution. The names and dosages of positive control substances used for each test bacterium are shown in each Table. Solvents and positive control groups were shared with other tests performed simultaneously. Cultivation was carried out at 37 ° C. for 48 hours. Toxicity was observed at 50.0 μg/plate (TA100, TA1535, TA1537) and 100 μg/plate(TA98, WP2 uvrA) without an S9 mix, and at 100 μg/plate(TA100, TA1535, TA98, TA1537) and 250 μg/plate(WP2 uvrA) with an S9 mix. The test chemical did not induce gene mutation in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA - in the presence and absence of rat liver activation system and hence the chemical is not likely to classify as a gene mutant in vitro. 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from J-check

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

Mutagenicity Testing of test Chemicals by using Chinese hamster lung(CHL/IU)cells

GLP compliance:

not specified

Type of assay:

other: in vitro gene mutation study in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

Chinese hamster lung(CHL/IU)cells

Details on mammalian cell type (if applicable):

CHL / IU cells derived from Chinese · hamster were used in the test within 10 years of thawing succession age

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top 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:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Since divinylbenzene dissolves the plastic bottom, we used a glass dish (6 cm in diameter) for culturing . 2 × 10 4 CHL / IU cells were plated in a dish containing 5 mL of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2). In the continuous treatment, test substances were added on day 3 of cell seeding and treated for 24 hours and 48 hours. In the short-term treatment, the cells were treated for 6 hours in the presence and absence of S9 mix on the third day of cell seeding, and after completion of the treatment, the cells were cultured for 18 hours with fresh culture medium.

Evaluation criteria:

cell proliferation rate measurement and the mitotic index

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

Chinese hamster lung(CHL/IU)cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Polyploidy was significant at 0.030 mg/mL(mid concentration) on continuous treatment. However, we concluded that divinylbenzene did not induce polyploidy since the frequency was low(1.88 %)and no significance was observed with a trend test.

Remarks on result:

other: Non mutagenic

Conclusions:

The test chemical did not induce chromosome aberrations in the Chinese hamster lung (CHL/IU) cells in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster lung (CHL/IU) cells with and without Rat liver, induced with phenobarbital and 5,6-benzoflavone activation system. The test chemical was studied at a dose level of 0, 0.015, 0.030, 0.060 mg/mL and 0, 0.0075, 0.015, 0.030 mg/mL (in the absence of S9) and 0, 0.015, 0.030, 0.060 mg/mL (in the presence of S9). 5 concentrations were set at the common ratio 2 (24 and 48 hours continuous treatment and brief treatment in the presence of S9 mix: 0.0075, 0.015, 0.030, 0.060, 0.12 mg / mL, brief treatment in the absence of S9 mix: 0.0038, 0.0075, 0.015, 0.030, 0.060 mg / mL). Polyploidy was significant at 0.030 mg/mL(mid concentration) on continuous treatment. However, we concluded that divinylbenzene did not induce polyploidy since the frequency was low(1.88 %)and no significance was observed with a trend test. Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster lung (CHL/IU) cells in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data available for the test chemical and read across chemicals was reviewed to determine the in- vitro gene toxicity of Divinylbenzene (CAS no 1321-74-0). The studies are as mentioned below:

Ames test

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA with and without Rat liver, induced with phenobarbital and 5,6-benzoflavone activation system. 0.1 mL of the test substance preparation solution, 0.5 mL of the phosphate buffer solution (0.5 mL of the S 9 mix in the S9 mix addition test) and 0.1 mL of the test bacterium solution were mixed in a small test tube and preincubated at 37 ° C. for 20 minutes, Add 2 mL of top agar and mix and sonicate it on a synthetic medium flat plate. As a control group, a solvent to be used or several kinds of positive control substance solutions was used in place of the test substance preparation solution. The names and dosages of positive control substances used for each test bacterium are shown in each Table. Solvents and positive control groups were shared with other tests performed simultaneously. Cultivation was carried out at 37 ° C. for 48 hours. Toxicity was observed at 50.0 μg/plate (TA100, TA1535, TA1537) and 100 μg/plate(TA98, WP2 uvrA) without an S9 mix, and at 100 μg/plate(TA100, TA1535, TA98, TA1537) and 250 μg/plate(WP2 uvrA) with an S9 mix. The test chemical did not induce gene mutation in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA - in the presence and absence of rat liver activation system and hence the chemical is not likely to classify as a gene mutant in vitro. 

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, TA98 and E. coli WP2 and WP2 uvrA- with and without Liver enzymes activation system. Ten ml of minimal agar medium (not containing test compound) was poured into a square Petri dish (9 x 9 cm) which is tilted at a slight angle. The agar was then allowed to solidify into a wedge-shaped layer by standing at room temperature. Meanwhile, a 1000-µg/ml mixture of test compound in agar was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100-mg/mI solution of test compound in dimethyl sulfoxide. When appropriate, water or dimethoxyethane was used instead of dimethyl sulfoxide. The cooled agar plates were then placed on a level surface, and an overlay of the 10 ml of agar containing the test compound was poured onto the plate to form a reversed wedge of agar on top of the first wedge. A concentration gradient of compound was produced by allowing the compound in the upper wedge to diffuse into the lower layer for 2 hr at room temperature. The concentration range in this plate is approximately 100 to 1000µg/ml. Three additional plates with concentration ranges of 10 to 100µg/ml, 1 to 10µg/ml, and 0.1 to 1µg/ml were prepared. A streaking device consisting of 10 sterile 50-µL pipets was dipped into suspensions of the 10 test strains and allowed to fill by capillary action. The pipets were then touched to the upper edge of the gradient and drawn across the plate. The study was performed in the presence and absence of liver enzyle activating system and the plates were incubated for 48 hrs at 37°C. The test chemical did not induce gene mutation in Salmonella typhimurium G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, TA98 and E. coli WP2 and WP2 uvrA- in the presence and absence of Liver enzymes activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation protocol using Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system. Preincubation was carried at 37°C for 20 mins followed by exposure period of 48 hrs at dose levels of 0.0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate. The test chemical failed to induce mutation in the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

 

Chromosome aberration:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster lung (CHL/IU) cells with and without Rat liver, induced with phenobarbital and 5,6-benzoflavone activation system. The test chemical was studied at a dose level of 0, 0.015, 0.030, 0.060 mg/mL and 0, 0.0075, 0.015, 0.030 mg/mL (in the absence of S9) and 0, 0.015, 0.030, 0.060 mg/mL (in the presence of S9). 5 concentrations were set at the common ratio 2 (24 and 48 hours continuous treatment and brief treatment in the presence of S9 mix: 0.0075, 0.015, 0.030, 0.060, 0.12 mg / mL, brief treatment in the absence of S9 mix: 0.0038, 0.0075, 0.015, 0.030, 0.060 mg / mL). Polyploidy was significant at 0.030 mg/mL(mid concentration) on continuous treatment. However, we concluded that divinylbenzene did not induce polyploidy since the frequency was low(1.88 %)and no significance was observed with a trend test. Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster lung (CHL/IU) cells in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro. 

In vitro mammalian chromosome aberration test was also performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 50-150µg/mL (in the absence of S9) and 25- 100µg/mL (in the presence of S9) using Chinese hamster ovary cells (CHO-W-B1). Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9. 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

 

In the same study, sister chromatid exchange test was performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 75-150µg/mL using Chinese hamster ovary cells (CHO-W-B1) both in the presence and absence of S9 metabolic activation system. 5-Bromodeoxyuridine (BrdUrd; 10 pM) was added 2 hr after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment periods were approximately 25 hr without S9 and 2 hr with S9. The total incubation time with BrdUrd was 25-26 hr, with colcemid (0.1µg/ml) present during the final 2-3 hr. immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield sufficient metaphase cells for analysis; supernatant medium was returned to appropriate flasks so that subsequent harvests could be made from the same cultures if necessary. Because all mitotic cells were removed in the initial harvest, cells collected during subsequent harvests had come into mitosis during the period between harvests and thus had been exposed to colcemid for an average of 4 hr. After 1-3 min treatment with hypotonic solution (75 mM KCl), cells were fixed in 3: 1 methano1: glacial acetic acid (V/V). For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with “dilute” Hoeschst 33258 (0.5µg/ml in Sorensen’s buffer, pH 6.8) and examined by fluorescence microscopy to assess cell cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25-26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis. After staining for 10 min in “concentrated” Hoechst 33258 (5µg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa. All slides were coded, and 50 cells per dose were scored from the three highest doses at which sufficient M2 cells were available. When cell cycle delay was noted, cell kinetics were recorded by classifying each of 100 metaphases as M1, M1+, or M2, i.e., having completed one (M1), two (M2), or between one and two (M1 +) cell cycles in BrdUrd. The test chemical did not induce an increase in the number of Sister chromatid exchanges in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the test chemical, Divinylbenzene (CAS no 1321-74-0) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the test chemical, Divinylbenzene (CAS no 1321-74-0) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.