1,4-divinylbenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical using Salmonella typhimurium both in the presence and absence of metabolic activation system. The test chemical did not induce gene mutation in Salmonella typhimurium and hence is not likely to classify as a gene mutant in vitro.

Chromosome aberration study:

In vitro mammalian chromosome aberration test study was performed to determine the mutagenic nature of the test chemical using Chinese hamster cell line both in the presence and absence of metabolic activation system. The test chemical did not induce chromosomal aberration in the Chinese hamster cell line 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

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of read across substances

Justification for type of information:

Weight of evidence approach based on structurally similar chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

WoE report is based on two in-vitro gene toxicity studies 1. and 2. Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

1. Histidine for Salmonella typhimurium strains and tryptophan for E. coli strains2. Histidine

Species / strain / cell type:

other: Salmonella typhimurium G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, TA98

Remarks:

1.

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli, other: WP2 and WP2 uvrA

Remarks:

1.

Additional strain / cell type characteristics:

other: WP2 (E. coli tryptophan) and WP2 uvrA (E. coli tryptophan with uvrA deletion).

Species / strain / cell type:

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

Remarks:

2.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Liver enzymes were prepared from adult male Fischer rats predosed with Aroclor 1254

Test concentrations with justification for top dose:

1. 0.1 – 1000 μg/mL2. 0.0, 3.3, 10.0, 33.0, 100.0, 333.0 μg/plate

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: DMSO, water or dimethoxyethane- Justification for choice of solvent/vehicle: Solubility2. - Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Remarks:

1.

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

other: Streptozotocin (without microsomal activation)

Untreated negative controls:

not specified

Remarks:

DMSO 2.

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene (2-AA), 4-Nitro-o-phenylenediamine (NOPD)

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in agar (gradient plate method)DURATION- Preincubation period: No data- Exposure duration: 48 hrs- Expression time (cells in growth medium): 48 hrs- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: Duplicate sets of plates were prepared: one set in which the test compound was in the bottom wedge and had to diffuse upward and one in which it was in the upper wedge andhad to diffuse downward.NUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data2. METHOD OF APPLICATION: preincubationDURATION- Preincubation period: 20 mins- Exposure duration: 48 hrs- Expression time (cells in growth medium): 48 hrs- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: TriplicateNUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1. Bacterial growth was observed along the streak line and the concentration range over which chemically induced mutant colonies are present is recorded.2. A dose related increase in the number of revertants was noted

Statistics:

No data

Species / strain:

S. typhimurium, other: G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, TA98

Remarks:

1.

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, other: WP2 and WP2 uvrA

Remarks:

1.

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:

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

Remarks:

2.

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

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce gene mutation in Salmonella typhimurium and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the test chemical and read across chemicals was reviewed to determine the in- vitro gene toxicity of 1,4-divinylbenzene (CAS no 105-06-6). The studies are as mentioned below:

Study 1:

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.

Study 2:

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.

Based on the data summarized, 1,4-divinylbenzene (CAS no 105-06-6) is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of read across substances

Justification for type of information:

Weight of evidence approach based on structurally similar chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as below

Principles of method if other than guideline:

WoE report is based on two in-vitro gene toxicity studies 1. In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical2. Sister Chromatid Exchange test was performed to determine the mutagenic nature of the test chemical.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster ovary cells (CHO-W-B1)

Remarks:

1.

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics- Properly maintained: No data available- Periodically checked for Mycoplasma contamination: No data available- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

mammalian cell line, other: Chinese hamster ovary cells (CHO-W-B1)

Remarks:

2.

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics- Properly maintained: No data available- Periodically checked for Mycoplasma contamination: No data available- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix consisted of 15 μl/ml liver homogenate (from male Sprague-Dawley rats, induced with Arocl or 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.

Test concentrations with justification for top dose:

1. 50-150 μg/mL (in the absence of S9) and 25- 100 μg/mL (in the presence of S9)2. 75-150 μg/mL

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: The chemical was dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference. Details are not available- Justification for choice of solvent/vehicle: Solubility2. - Vehicle(s)/solvent(s) used: The chemical was dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference. Details are not available- Justification for choice of solvent/vehicle: Solubility

Untreated negative controls:

not specified

Remarks:

1.

Negative solvent / vehicle controls:

yes

Remarks:

water, dimethyl sulfoxide (DMSO), ethanol, or acetone (Details are not specified)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Triethylenemelamine

Untreated negative controls:

not specified

Remarks:

2.

Negative solvent / vehicle controls:

yes

Remarks:

Water, DMSO, ethanol or acetone (Details are not available)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in mediumDURATION- Preincubation period: No data- Exposure duration: Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9.- Expression time (cells in growth medium): 18-26 hrs during the delayed harvest time- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): the cell harvest time for the aberration test was 8-12 hr after the beginning of treatment. This yielded cells in their first mitosis. Depending on the amount of delay seen in the SCE test, later harvest times, eg, 18-26 hr, were used to allow delayed cells to reach mitosis.SELECTION AGENT (mutation assays): GiemsaSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: No dataNUMBER OF CELLS EVALUATED: 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysisDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data2.METHOD OF APPLICATION: in mediumDURATION- Preincubation period: No data- Exposure duration: The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9.- Expression time (cells in growth medium): 25-26 hrs- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): After staining for 10 min in “concentrated” Hoechst 33258 (5 pg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in GiemsaSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: No dataNUMBER OF CELLS EVALUATED: 50 cells per dose were scored from the three highest dosesDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: Cells were collected by mitotic shake off for evaluation

Evaluation criteria:

1. 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 endoreduplications were recorded but were not included in the totals. We did not score aberrations in polyploidy cells but used metaphases with 19-23 chromosomes (the modal number being 21).2.An increase in sister chromatid exchange was noted

Statistics:

1. Linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et al. The Pvalues were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.2.Linear regression test (trend test) of SCEs per chromosome vs the log of the dose. For individual doses, absolute increases in SCEs per chromosome of 20% or more over the solvent control were considered significant.

Species / strain:

mammalian cell line, other: Chinese hamster ovary cells (CHO-W-B1)

Remarks:

1.

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:

mammalian cell line, other: Chinese hamster ovary cells (CHO-W-B1)

Remarks:

2.

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

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosomal aberration in the Chinese hamster cell line and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Chromosome aberration:

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 appoximately 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 bemade 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, 1,4-divinylbenzene (CAS no 105-06-6) 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene toxicity – in-vitro

Data available for the test chemical and read across chemicals was reviewed to determine the in- vitro gene toxicity of 1,4-divinylbenzene (CAS no 105-06-6). The studies are as mentioned below:

Ames test

Study 1:

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.

Study 2:

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 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 appoximately 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 bemade 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, 1,4-divinylbenzene (CAS no 105-06-6) 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, 1,4-divinylbenzene (CAS no 105-06-6) 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.