Registration Dossier
Registration Dossier
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EC number: 219-110-7 | CAS number: 2362-14-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Negative with and without metabolic activation in S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA (bacterial reverse mutation assay); OECD 471
Negative with and without metabolic activation in L5178Y TK+/- mouse lymphoma cells (mammalian cell gene mutation assay); OECD 476
Positive for the induction of structural chromosome aberrations in CHO cells in the S9 activated 4-hour exposure group and in the non-activated 20-hour exposure group (mammalian chromosome aberration test); OECD 473
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- bacteria, other: Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 micrograms per plate with and w/out S9 activation (Preliminary toxicity and initial mutagenicity assay)
5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate (Independent repeat/confirmatory mutagenicity assay) - Vehicle / solvent:
- DMSO (CAS No. 67-68-5); from Fisher Scientific
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- for all strains; 1.0 µg/plate for all S. typhimurium; 10 µg/plate E. coli
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- TA98 - Without S9 activation; 1.0 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide
- Remarks:
- TA100, TA1535 - Without S9 activation; 1.0 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537 - Without S9 activation; 75 µg/plate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- WP2 uvrA - Without S9 activation; 1000 µg/plate
- Details on test system and experimental conditions:
- Preliminary Toxicity/Initial Mutagenicity Assay: The preliminary toxicity/initial mutagenicity assay was used to establish the dose range over which the test article would be assayed and to provide a preliminary mutagenicity evaluation. A vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9.
Confirmatory Mutagenicity Assay: The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Six dose levels of test article along with vehicle control and appropriate positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.
Plating and Scoring Procedures: The test system was exposed to the test article via the plate incorporation method. On the day of its use, minimal top agar was melted and supplemented. Top agar, not used with S9 or Sham mix, was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionised water produced by the Milli Q Reagent Water System. Bottom agar was supplemented Vogel Bonner minimal medium E.
Each plate was labelled with a code system that identified the test article, test phase, dose level, tester strain and activation.
Test article dilutions were prepared immediately before use. One half (0.5) millilitre of S9 or Sham mix, 100 µL of tester strain and 50 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45 ± 2 °C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 ± °C. Plates that were not counted immediately following the incubation period were stored at 2 - 8 °C until colony counting could be conducted.
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation was scored relative to the vehicle control plate.
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automatic colony counting were counted manually. - Evaluation criteria:
- For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.
For the test article to be evaluated positive, it must have caused a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value. - Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Generally observed beginning at 150, 500 or 1500 µg/plate in the confirmatory assay.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- In the preliminary toxicity/initial mutagenicity assay (Experiments B1 and B2), the doses tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 µg/plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1800 µg/plate. Toxicity was generally observed beginning at 200, 600 or 1800 µg/plate. Due to an unacceptable vehicle control value, tester strain WP2 uvrA in the absence of S9 activation was not evaluated in Experiment B1 but was successfully retested in Experiment B2. Based on the preliminary toxicity findings the doses tested for the confirmatory mutagenicity assay were 5, 15, 50, 150, 500 and 1500 µg/plate without metabolic activation and 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation.
For the Confirmatory Mutagenicity Assay (Experiment B3), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 5000 µg/plate with S9 activation. Toxicity was generally observed beginning at 150, 500 or 150 µg/plate. - Conclusions:
- Under the conditions of this study, the test material was negative in both the presence and absence of metabolic activation.
- Executive summary:
The potential of the test material to cause genetic toxicity was investigated in a bacterial reverse mutation assay conducted in accordance with the standardised guideline OECD 471 under GLP conditions.
Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA were exposed to the test material in DMSO via the plate incorporation method at dose levels of 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 micrograms per plate with and without S9 activation in a preliminary toxicity and initial mutagenicity assay. The strains were then exposed to 5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate in an independent repeat/confirmatory mutagenicity assay.
In the preliminary toxicity/initial mutagenicity assay (Experiments B1 and B2) no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1800 µg/plate. Toxicity was generally observed beginning at 200, 600 or 1800 µg/plate. Due to an unacceptable vehicle control value, tester strain WP2 uvrA in the absence of S9 activation was not evaluated in Experiment B1 but was successfully retested in Experiment B2.
For the confirmatory mutagenicity assay (Experiment B3), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 5000 µg/plate with S9 activation. Toxicity was generally observed beginning at 150, 500 or 150 µg/plate. All criteria for a valid study were met.
Under the conditions of this study, the test material was negative in both the presence and absence of metabolic activation.
- Endpoint:
- in vitro gene mutation 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
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- TK+/-
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9 prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor-1254, 500 mg/kg, five days prior to sacrifice
- Test concentrations with justification for top dose:
- 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 3000 µg/mL with and without S9 activation (Preliminary assay)
5, 7.5, 10, 12.5, 15 and 17.5 µg/mL (Mutagenicity assay: 4-hour exposure without S9 activation)
5, 7.5, 10, 12.5 and 15 µg/mL (Concentrations chosen for cloning; Mutagenicity assay: 4-hour exposure without S9 activation
15, 20, 25, 30 35 and 40 µg/mL (Mutagenicity assay: 4-hour exposure with S9 activation)
15, 20, 25, 30 and 35 µg/mL (Concentrations chosen for cloning; Mutagenicity assay: 4-hour exposure with S9 activation) - Vehicle / solvent:
- DMSO (CAS No. 67-68-5); from Fisher Scientific
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with S9 activation; 1.0 and 1.5 microg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without S9 activation; 10 and 20 microg/mL
- Details on test system and experimental conditions:
- Description of test procedure: The preliminary toxicity assay was used to establish the optimal dose levels for the mutagenesis assay. L5178Y cells were exposed to the solvent alone and nine concentrations of test article ranging from 0.5 to 3000 µg/mL in both the absence and presence of S9-activation with a 4 hour exposure. Cell population density was determined 24 and 48 hours after the initial exposure to the test article. The cultures were adjusted to 3 x 10⁵ cells/mL after 24 hours only. Toxicity was measured as suspension growth of the treated cultures relative to the growth of the solvent control cultures after 48 hours.
The mutagenesis assay was used to evaluate the mutagenic potential of the test article (with and without activation with a 4 hour exposure) by exposing L5178Y mouse lymphoma cells to the solvent alone and at least eight concentrations of the test article in duplicate in both the presence and absence of S9. The positive controls with and without metabolic activation were tested concurrently.
Treatment of target cells: Treatment was carried out in conical tubes by combining 6 x 10⁶ L5178Y TK+/- cells, F0P medium or S9 activation mixture, and 100 µL dosing solution of test or control article in solvent or solvent alone in a total volume of 10 mL. The positive controls were treated with MMS (at final concentrations in treatment medium of 10 and 20 µg/mL in the absence of S9 activation) and 7,12-DMBA (at final concentrations in treatment medium of 1.0 and 1.5 µg/mL in the presence of S9 activation). Treatment tubes were gassed with 5 ± 1 % CO₂ in air, capped tightly, and incubated with mechanical mixing for 4 hours at 37 ± 1 °C. The preparation and addition of the test article dosing solutions were carried out under amber lighting and the cells were incubated in the dark during the exposure period. After the treatment period, the cells were washed twice and resuspended in F10P medium, gassed with 5 ± 1 % CO₂ in air and placed on the roller drum apparatus at 37 ± 1 °C.
Expression of the mutant phenotype: For expression of the mutant phenotype, the cultures were counted and adjusted to 3 x 10⁵ cells/mL at approximately 24 and 48 hours after treatment in 20 and 10 mL total volume, respectively. Cultures with less than 3 x 10⁵ cells/mL were not adjusted. For expression of the TK-/- cells, cells were placed in cloning medium (CM). Two flasks per culture to be cloned were labelled with the test article concentration, activation condition, and either TFT (trifluorothymidine, the selective agent) or VC (viable count). Each flask was pre-warmed to 37 ± 1 °C, filled with 100 mL CM, and placed in an incubator shaker at 37 ± 1 °C until used. The cells were centrifuged at 1000 rpm for 10 minutes and the supernatant was decanted. The cells were then diluted in CM to concentrations of 3 x 10⁶ cells/100 mL CM for the TFT flask and 600 cells/100 mL CM for the VC flask. After the dilution, 1.0 mL of stock solution of TFT was added to the TFT flask (final concentration of 3 µg/mL) and both this flask and the VC flask were placed on the shaker at 125 rpm and 37 ± 1 °C for 15 minutes. After 15 minutes, the flasks were removed and 33 mL of the cell suspension was divided equally into each of three appropriately labelled Petri dishes. To accelerate the gelling process, the plates were placed in cold storage (approximately 4 °C) for approximately 30 minutes. The plates were then incubated at 37 ± 1 °C in a humidified 5 ± 1 % CO₂ atmosphere for 10 - 14 days.
Scoring procedures: After incubation, the VC plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were counted for each culture with greater than or equal to 10 % total relative growth. The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test article-treated cultures were determined over a range of approximately 0.2 to 1.1 mm. - Evaluation criteria:
- The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency (number of mutants per 10⁶ surviving cells) was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding VC plates and multiplying by the dilution factor (2 x 10^-4) then multiplying by 10⁶. In evaluation of the data, increases in mutant frequencies that occurred only at highly toxic concentrations (i.e., less than 10 % total growth) were not considered biologically relevant. The following criteria are presented as a guide to interpretation of the data: (1) A result was considered positive if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10 % or greater total growth exhibited mutant frequencies of greater than or equal to 100 mutants per 10⁶ clonable cells over the background level. (2) A result was considered equivocal if the mutant frequency in treated cultures was between 55 and 99 mutants per 10⁶ clonable cells over the background level. (3) A result was considered negative if the mutant frequency in treated cultures was fewer than 55 mutants per 10⁶ clonable cells over the background level.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The test article was soluble in DMSO at the maximum concentration tested.
The maximum concentration tested in the preliminary toxicity assay was 3000 µg/mL (10mM). Visible precipitate was present at concentrations greater than or equal to 150 µg/mL in treatment medium. No visible precipitate was present at concentrations of less than or equal to 50 µg/mL in treatment medium. The osmolality of the solvent control was 449 mmol/kg and the osmolality of the highest soluble dose, 50 µg/mL, was 410 mmol/kg. Suspension growth relative to the solvent controls was 0 % at 50 µg/mL with and without S9 activation. Based on the results of the toxicity test, the doses chosen for the mutagenesis assay ranged from 5.0 to 50 µg/mL for both the non-activated and S9 activated cultures.
In the first mutation assay, no visible precipitate was present at any dose level in treatment medium. In the non-activated system, cultures treated with concentrations of 5.0, 7.5, 10, 12.5 and 15 µg/mL were cloned and produced a range in suspension growth of 10 to 101 %. In the S9-activated system, cultures treated with concentrations of 15, 20, 25, 30 and 35 µg/mL were cloned and produced a range in suspension growth of 18 to 112 %. No cloned cultures exhibited mutant frequencies that were at least 55 mutants per 10⁶ clonable cells over that of the solvent control. A dose-response trend was not observed in the non-activated or S9-activated systems. The total growths ranged from 10 to 99 % for the non-activated cultures at concentrations of 5.0 to 15 µg/mL and 15 to 101 % for the S9-activated cultures at concentrations of 15 to 35 µg/mL. The TFT-resistant colonies for the positive and solvent control cultures were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS positive control yielded the expected increase in small colonies, verifying the adequacy of the methods used to detect small colony mutants.
All criteria for a valid test were met. - Conclusions:
- Under the conditions of this study, the test material was considered to be negative with and without metabolic activation in the L5178Y TK+/- mouse lymphoma mutagenesis assay.
- Executive summary:
The genotoxicity potential of the test material was investigated in a mammalian cell gene mutation assay conducted in accordance with the standardised guideline OECD 476 under GLP conditions.
Mouse lymphoma L5178Y TK+/- cells were exposed to the test material in DMSO at the following concentrations: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 3000 µg/mL with and without S9 activation in a preliminary assay; 5, 7.5, 10, 12.5, 15 and 17.5 µg/mL in the 4 hour mutagenicity assay without S9 activation; and 15, 20, 25, 30 35 and 40 µg/mL in the 4 hour mutagenicity assay with S9 activation.
For expression of the mutant phenotype, the cultures were counted and adjusted to 3 x 10⁵ cells/mL at approximately 24 and 48 hours after treatment in 20 and 10 mL total volume, respectively. Following the appropriate treatment, the cells were incubated for 10 to 14 days.
In the first mutation assay, no visible precipitate was present at any dose level in treatment medium. In the non-activated system, cultures treated with concentrations of 5.0, 7.5, 10, 12.5 and 15 µg/mL were cloned and produced a range in suspension growth of 10 to 101 %. In the S9-activated system, cultures treated with concentrations of 15, 20, 25, 30 and 35 µg/mL were cloned and produced a range in suspension growth of 18 to 112 %. No cloned cultures exhibited mutant frequencies that were at least 55 mutants per 10⁶ clonable cells over that of the solvent control. A dose-response trend was not observed in the non-activated or S9-activated systems. The total growths ranged from 10 to 99 % for the non-activated cultures at concentrations of 5.0 to 15 µg/mL and 15 to 101 % for the S9-activated cultures at concentrations of 15 to 35 µg/mL. The TFT-resistant colonies for the positive and solvent control cultures were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS positive control yielded the expected increase in small colonies, verifying the adequacy of the methods used to detect small colony mutants. Therefore all criteria for a valid test were met.
Under the conditions of this study, the test material was considered to be negative with and without metabolic activation in the L5178Y TK+/- mouse lymphoma mutagenesis assay.
- 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
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- 0.296, 0.888, 2.96, 8.88, 29.6, 88.8, 296, 888 and 2960 µg/mL (Preliminary toxicity assay)
2.5, 5, 10, 12.5, 15, 17.5, 20, and 25 µg/mL (Chromosome Aberration Assay - 4-hour treatment without S9 activation)
1.25, 2.5, 5, 10, 12.5, 15, 17.5, 20, and 25 µg/mL (Chromosome Aberration Assay – 4 hour treatment with S9 activation and 20-hour without S9 activation) - Vehicle / solvent:
- DMSO (CAS No. 67-68-5); from Fisher Scientific
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with activation; 10 and 20 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without activation; 0.1 and 0.2 µg/mL
- Details on test system and experimental conditions:
- Description of test procedure: A preliminary toxicity assay was performed for the purpose of selecting dose levels for the chromosome aberration assay and consisted of an evaluation of test article effect on cell growth. The osmolality and pH (using test tape) of the highest concentration of test article dosing solution in treatment medium was measured. CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 0.296 µg/mL to 2960 µg/mL in the absence and presence of an S9 reaction mixture. The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9. At completion of the 4 hour exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), re-fed with 5 mL complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. At 20 hours after the initiation of treatment the cells were harvested by trypsinisation and counted using a Coulter counter. The presence of test article precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.
For the chromosome aberration assay, CHO cells were seeded for each treatment condition at approximately 5 x 10⁵ cells/25 cm² flask and were incubated at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO₂ in air for 16 - 24 hours. Treatment was carried out by refeeding duplicate flasks with 5 mL of appropriately supplemented complete medium for the non-activated study or 5 mL S9 reaction mixture for the S9-activated study, to which was added 50 µL of dosing solution of test article in solvent or solvent alone. The osmolality and pH (using test tape) of the highest concentration of test article dosing solution in treatment medium was measured.
The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9 at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO₂ in air. At completion of exposure for the 4 hour exposure groups, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), re-fed with complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. Two hours prior to cell harvest, Colcemid® was added to duplicate flasks for each treatment condition at a final concentration of 0.1 µg/mL and the flasks were returned to the incubator until cell collection.
Two hours after the addition of Colcemid, metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinisation. The cells were collected approximately 20 hours after initiation of treatment by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 2-4 mL 0.075 M potassium chloride (KCl) and allowed to stand at room temperature for 4-8 minutes. The cells were collected by centrifugation, the supernatant aspirated and the cells fixed with two washes of approximately 2 mL Carnoy's fixative (methanol:glacial acetic acid, 3:1, v/v). The cells were stored overnight or longer in fixative at approximately 2-8 °C.
To prepare slides, the fixed cells were centrifuged at approximately 800 rpm for 5 minutes, the supernatant was aspirated, and fresh fixative was added. After additional centrifugation (at approximately 800 rpm for 5 minutes) the supernatant fluid was decanted and the cells resuspended to opalescence in fresh fixative. A sufficient amount of cell suspension was dropped onto the centre of a glass slide and allowed to air dry. The dried slides were appropriately identified, stained with 5 % Giemsa, air dried and permanently mounted.
A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems. After cell harvest an aliquot of the cell suspension was removed from each culture and counted using a Coulter counter. The presence of test article precipitate was assessed using the unaided eye. Cell viability was determined by trypan blue dye exclusion. The cell counts and percent viability were used to determine cell growth inhibition relative to the solvent control.
Evaluation of metaphase cells:
The selection of dose levels for analysis of chromosome aberrations in CHO cells was based on toxicity. In the S9 activated 4 hour exposure group and in the non-activated 4 hour exposure group, the highest dose evaluated for chromosome aberrations was the lowest dose with at least 50 % reduction in cell growth. In the non-activated 20-hour continuous exposure group, the highest dose evaluated was the lowest dose with at least 50 % reduction in mitotic index. Two additional lower dose levels were also evaluated in each harvest.
To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphase spreads that were examined and scored per duplicate flask was reduced if the percentage of aberrant cells reached a statistically significant level before 100 cells were scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverised chromosome(s), pulverised cells and severely damaged cells (greater than or equal to 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY coordinates for each cell with chromosomal aberrations were recorded using a calibrated microscope stage. Polyploid and endoreduplicated cells were evaluated from each treatment flask per 100 metaphase cells scored.
Plates/test: Samples were run in duplicate, with and without metabolic activation.
Activation system: Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. - Evaluation criteria:
- The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
- Statistics:
- Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's exact test at any test article dose level, the Cochran Armitage test was used to measure dose-responsiveness. All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article is considered to induce a positive response when the percentage of cells with aberrations is increased in a dose responsive manner with one or more concentrations being statistically significant (p less than or equal to 0.05). Test articles not demonstrating a statistically significant increase in aberrations are concluded to be negative.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- (4 hour exposure group)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 55 % cell growh inhibition at 15 µg/mL and 100 % at 17.5 µg/mL and higher
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- (4 hour exposure group)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 61 % cell growth inhibition at 25 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- (20 hour exposure group)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 55 % cell growth inhibition at 25 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- In the preliminary toxicity test, DMBPC was soluble in treatment medium at dose levels greater than or equal to 88.8 µg/mL. Visible precipitate was observed in treatment medium at dose levels greater than or equal to 296 µg/mL. The osmolality in treatment medium of the highest concentration tested, 2960 µg/mL, was 377 mmol/kg. The osmolality in treatment medium of the lowest precipitating concentration tested, 296 µg/mL, was 398 mmol/kg. The osmolality in treatment medium of the highest soluble concentration tested, 88.8 µg/mL, was 414 mmol/kg. The osmolality of the solvent (DMSO) in treatment medium was 416 mmol/kg. The osmolality of the DMBPC concentrations in treatment medium were acceptable because they did not exceed the osmolality of the solvent by more than 20 %. The pH of the highest concentration of test article in treatment medium was 7.5. Cell growth inhibition relative to the solvent control was 100 % at 2960 µg/mL, the highest concentration tested in both the 4 and 20 hour non-activated exposure groups, and in the S9 activated 4 hour exposure group. Based on the results of the toxicity study, the dose levels selected for testing in the chromosome aberration assay were 2.5, 5, 10, 12.5, 15, 17.5, 20 and 25 µg/mL in the 4-hour non-activated assay; and 1.25, 2.5, 5 10, 12.5, 15, 17.5, 20 and 25 for the 4-hour treatment with S9 activation and 20-hour without S9 activation.
In the chromosome aberration assay, DMBPC was soluble in treatment medium at all dose levels tested. The osmolality in treatment medium of the highest concentration tested, 25 µg/mL, was 414 mmol/kg. The osmolality of the solvent (DMSO) in treatment medium was 428 mmol/kg. The osmolality of the DMBPC concentrations in treatment medium were acceptable because they did not exceed the osmolality of the solvent by more than 20 %. The pH of the highest concentration of test article in treatment medium was approximately 7.5.
Toxicity of DMBPC in CHO cells treated for 4-hours without metabolic (S9) activation was 61 % at 25 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest test concentration evaluated for chromosome aberrations (25 µg/mL) was 10 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 10 and 25 µg/mL. The percentage of cells with structural and numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control (p greater than 0.05, Fisher’s Exact test). The percentage of structurally damaged cells in the MMC (positive control) treatment group (16.0 %) was found to be statistically significant.
Toxicity of DMBPC in CHO cells when treated for 4-hours with S9 activation was 55 % at 15 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest test concentration evaluated for chromosome aberrations (15 µg/mL) was 69 % reduced relative to the solvent control.
The dose levels selected for microscopic analysis were 2.5, 5.0 and 15 µg/mL. The percentage of cells with structural aberrations in the test article-treated group was statistically increased above that of the solvent control at all concentrations evaluated (p less than or equal to 0.05 and 0.01, Fisher’s Exact test). The Cochran-Armitage test was also positive for a dose response. However, the percentage of cells with structural aberrations (5.5 %) in the DMBPC-treated groups at 2.5 µg/mL was within the historic solvent control range of 0.0 to 6.5 %. Therefore, this would not be considered biologically significant. The percentage of structurally aberrant cells at concentrations of 5 and 15 µg/mL, 9.0 and 8.5 %, respectively, fall just outside the historical solvent control range and this is considered to be positive. The percentage of cells with numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control. The percentage of structurally damaged cells in the positive control (CP) group (40.0 %) was statistically significant.
In the absence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20-hour exposure group were evaluated for chromosome aberrations. Toxicity of DMBPC in CHO cells was 31 % at 17.5 µg/mL, the highest test concentration evaluated for chromosome aberrations in the non-activated 20-hour continuous exposure. The mitotic index at the highest test concentration evaluated for chromosome aberrations (17.5 µg/mL) was 68 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 10 and 17.5 µg/mL. The percentage of cells with structural aberrations in the test article-treated groups was significantly increased above that of the solvent control at 17.5 µg/mL (Fisher’s exact test). The Cochran-Armitage test was negative for a dose response. The percentage of cells with structural aberrations in the DMBPC-treated at 17.5 µg/mL group (8.0 %) was outside the historical solvent control range of 0.0 to 6.0 % and would be considered positive for the induction of structural chromosome aberrations. The percentage of cells with numerical aberrations in the DMBPC-treated groups was not statistically significantly increased above that of the solvent controls at any dose level. The percentage of structurally damaged cells in the positive control (MMC) group (21.0 %) was statistically significant.
The positive and solvent controls fulfilled the requirements for a valid test. - Conclusions:
- Based on the findings of this study, DMBPC was concluded to be positive for the induction of structural chromosome aberrations in CHO cells in the S9 activated 4-hour exposure group and in the non-activated 20-hour exposure group. DMBPC was concluded to be negative for the induction of numerical chromosome aberrations in CHO cells.
- Executive summary:
The genotoxicity potential of the test material was investigated in an in vitro mammalian chromosome aberration test conducted in accordance with the standardised guideline OECD 473 under GLP conditions.
Chinese hamster ovary cells (CHO-K1) were exposed to the test material in DMSO at the following concentrations: 0.296, 0.888, 2.96, 8.88, 29.6, 88.8, 296, 888 and 2960 µg/mL in the preliminary toxicity assay; 2.5, 5, 10, 12.5, 15, 17.5, 20, and 25 µg/mL in the 4 hour chromosome aberration assay without S9 activation; and 1.25, 2.5, 5, 10, 12.5, 15, 17.5, 20, and 25 µg/mL in the 4 hour chromosome aberration assay with S9 activation and 20-hour assay without S9 activation.
The cells were treated for 4 hours with and without S9, and continuously for 20 hours without S9 at 37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO₂ in air. At completion of exposure for the 4 hour exposure groups, the treatment medium was removed, the cells washed, re-fed with complete medium and returned to the incubator for a total of 20 hours from the initiation of treatment. Two hours prior to cell harvest, Colcemid® was added and the flasks were returned to the incubator until cell collection. Two hours after the addition of Colcemid, metaphase cells were harvested for both the non-activated and S9 activated studies by trypsinisation. Following collection, the cells were fixed and slides prepared. Metaphase cells were then evaluated. A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems.
Toxicity of DMBPC in CHO cells treated for 4-hours without metabolic (S9) activation was 61 % at 25 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest test concentration evaluated for chromosome aberrations (25 µg/mL) was 10 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 10 and 25 µg/mL. The percentage of cells with structural and numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control. The percentage of structurally damaged cells in the positive control treatment group (16.0 %) was found to be statistically significant.
Toxicity of DMBPC in CHO cells when treated for 4-hours with S9 activation was 55 % at 15 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest test concentration evaluated for chromosome aberrations (15 µg/mL) was 69 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 5.0 and 15 µg/mL. The percentage of cells with structural aberrations in the test article-treated group was statistically increased above that of the solvent control at all concentrations evaluated. However, the percentage of cells with structural aberrations (5.5 %) in the DMBPC-treated groups at 2.5 µg/mL was within the historic solvent control range of 0.0 to 6.5 %. Therefore, this would not be considered biologically significant. The percentage of structurally aberrant cells at concentrations of 5 and 15 µg/mL, 9.0 and 8.5 %, respectively, fall just outside the historical solvent control range and this is considered to be positive. The percentage of cells with numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control. The percentage of structurally damaged cells in the positive control group (40.0 %) was statistically significant.
In the absence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20-hour exposure group were evaluated for chromosome aberrations. Toxicity of DMBPC in CHO cells was 31 % at 17.5 µg/mL, the highest test concentration evaluated for chromosome aberrations in the non-activated 20-hour continuous exposure. The mitotic index at the highest test concentration evaluated for chromosome aberrations (17.5 µg/mL) was 68 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 10 and 17.5 µg/mL. The percentage of cells with structural aberrations in the test article-treated groups was significantly increased above that of the solvent control at 17.5 µg/mL.The percentage of cells with structural aberrations in the DMBPC-treated at 17.5 µg/mL group (8.0 %) was outside the historical solvent control range of 0.0 to 6.0 % and would be considered positive for the induction of structural chromosome aberrations. The percentage of cells with numerical aberrations in the DMBPC-treated groups was not statistically significantly increased above that of the solvent controls at any dose level. The percentage of structurally damaged cells in the positive control group (21.0 %) was statistically significant.
The positive and solvent controls fulfilled the requirements for a valid test.
Based on the findings of this study, DMBPC was concluded to be positive for the induction of structural chromosome aberrations in CHO cells in the S9 activated 4-hour exposure group and in the non-activated 20-hour exposure group. DMBPC was concluded to be negative for the induction of numerical chromosome aberrations in CHO cells.
Referenceopen allclose all
Initial Mutagenicity Assay (without activation)
Mean Number of Revertants Per Plate
Dose (µg/plate) | TA98 | TA100 | TA1535 | TA1537 | WP2uvrA |
Vehicle (DMSO) | 14 ± 1 | 162 ± 1 | 12 ± 1 | 5 ± 2 | 12 ± 1 |
2.5 | 9 ± 2 | 156 ± 15 | 10 ± 6 | 4 ± 1 | 9 ± 4 |
7.5 | 9 ± 3 | 169 ± 2 | 8 ± 2 | 3 ± 1 | 8 ± 1 |
25 | 11 ± 3 | 160 ± 18 | 10 ± 0 | 9 ± 1 | 10 ± 1 |
75 | 10 ± 1 | 112 ±3 | 11 ± 1 | 5 ± 4 | 7 ± 3 |
200 | 7 ± 2 | 87 ± 6 | 12 ± 4 | 2 ± 2 | 5 ± 1 |
600 | 4 ± 1 | 30 ± 2 | 5 ± 1 | 1 ± 1 | 2 ± 1 |
1800 | 1 ± 1 | 0 ± 0 | 1 ± 0 | 0 ± 0 | 1 ± 0 |
5000 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0 ± 0 |
Positive Control | 169 ± 25 | 747 ± 148 | 207 ± 0 | 453 ± 42 | 93 ± 1 |
Initial Mutagenicity Assay (with activation)
Mean Number of Revertants Per Plate
Dose (µg/plate) | TA98 | TA100 | TA1535 | TA1537 | WP2uvrA |
Vehicle (DMSO) | 12 ± 4 | 138 ± 24 | 9 ± 1 | 4 ± 1 | 10 ± 0 |
2.5 | 11 ± 0 | 176 ± 11 | 11 ± 4 | 4 ± 2 | 10 ± 5 |
7.5 | 12 ± 3 | 151 ± 5 | 9 ± 1 | 4 ± 3 | 8 ± -- |
25 | 13 ± 4 | 160 ± 35 | 9 ± 5 | 3 ± 2 | 8 ± 1 |
75 | 10 ± 0 | 154 ± 17 | 7 ± 2 | 5 ± 2 | 8 ± 1 |
200 | 6 ± 0 | 123 ± 14 | 8 ± 4 | 7 ± 1 | 8 ± 0 |
600 | 2 ± 0 | 100 ±18 | 7 ± 1 | 2 ± 0 | 5 ± 4 |
1800 | 2 ± 1 | 16 ± 7 | 3 ± 1 | 0 ± 0 | 0 ± 0 |
5000 | 0 ± 0 | 2 ± 1 | 0 ± 0 | 0 ± 0 | 0 ± 0 |
Positive Control | 1102 ± 265 | 1205 ± 39 | 141 ± 24 | 130 ± 21 | 227 ± 133 |
Confirmatory Mutagenicity Assay (without activation)
Mean Number of Revertants Per Plate
Dose (µg/plate) | TA98 | TA100 | TA1535 | TA1537 | WP2uvrA |
Vehicle (DMSO) | 12 ± 3 | 155 ± 15 | 12 ± 5 | 4 ± 2 | 11 ± 2 |
5.0 | 10 ± 1 | 163 ± 5 | 14 ± 1 | 5 ± 2 | 10 ± 1 |
15 | 13 ± 3 | 204 ± 10 | 9 ± 4 | 4 ± 1 | 11 ± 1 |
50 | 8 ± 1 | 106 ±9 | 12 ± 1 | 5 ± 3 | 9 ± 1 |
150 | 6 ± 2 | 36 ± 3 | 6 ± 1 | 2 ± 1 | 6 ± 3 |
500 | 1 ± 1 | 1 ± 2 | 2 ± 1 | 0 ± 0 | 0 ± 1 |
1500 | 0 ± 1 | 0 ± 0 | 0 ± 0 | 0 ± 0 | 0 ± 1 |
Positive Control | 87 ± 15 | 571 ± 21 | 135 ± 28 | 476 ± 145 | 70 ± 5 |
Confirmatory Mutagenicity Assay (with activation)
Mean Number of Revertants Per Plate
Dose (µg/plate) | TA98 | TA100 | TA1535 | TA1537 | WP2uvrA |
Vehicle (DMSO) | 14 ± 3 | 133 ± 21 | 6 ± 1 | 5 ± 2 | 11 ± |
15 | 14 ± 3 | 142 ± 6 | 5 ± 1 | 4 ± 2 | 11 ± 2 |
50 | 14 ± 3 | 140 ±21 | 7 ± 2 | 6 ± 1 | 11 ± 1 |
150 | 10 ± 1 | 79 ± 8 | 5 ± 1 | 3 ± 1 | 9 ± 2 |
500 | 3 ± 2 | 43 ± 2 | 4 ± 1 | 2 ± 1 | 4 ± 2 |
1500 | 1 ± 1 | 5 ± 1 | 1 ± 1 | 0 ± 1 | 1 ± 1 |
5000 | 0 ± 0 | 0 ± 1 | 0 ± 1 | 0 ± 0 | 0 ± 0 |
Positive Control | 1578 ± 365 | 1691 ± 552 | 122 ± 13 | 227 ± 21 | 138 ± 53 |
Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with DMBPC in the Absence of Exogenous Metabolic Activation Initial Assay (4-hour exposure)
Dose Level (µg/mL) | Rep | TFT Colonies | VC Colonies | Mutant Freq.a | Induced Mutant Freq.b | % Total Growthc | ||||||
Counts | Mean | Counts | Mean | |||||||||
0 (solvent) | 1 | 52 | 27 | 55 | 45 ± 13 | 168 | 188 | 183 | 180 ± 8 | 50 | -- | -- |
0 (solvent) | 2 | 31 | 50 | 59 | 47 ± 12 | 152 | 157 | 152 | 154 ± 2 | 61 | -- | -- |
Mean Solvent Mutant Frequency = 55 | ||||||||||||
5 | A | 31 | 25 | 22 | 26 ± 4 | 140 | 123 | 157 | 140 ± 14 | 37 | -18 | 84 |
5 | B | 25 | 44 | 68 | 46 ± 18 | 155 | 150 | 171 | 159 ± 9 | 58 | 2 | 95 |
7.5 | A | 32 | 37 | 40 | 36 ± 13 | 135 | 125 | 168 | 143 ± 18 | 51 | -4 | 75 |
7.5 | B | 52 | 25 | 47 | 41 ± 12 | 148 | 131 | 117 | 132 ± 13 | 63 | 7 | 80 |
10 | A | 48 | 33 | 50 | 44 ± 8 | 131 | 136 | 188 | 152 ± 26 | 58 | 2 | 56 |
10 | B | 55 | 28 | 55 | 46 ± 13 | 191 | 202 | 150 | 181 ± 22 | 51 | -4 | 99 |
12.5 | A | 20 | 44 | 45 | 36 ± 12 | 142 | 175 | 168 | 162 ± 14 | 45 | -10 | 33 |
12.5 | B | 24 | 14 | 33 | 24 ± 8 | 151 | 138 | 143 | 144 ± 5 | 33 | -22 | 49 |
15 | A | ++ |
|
|
|
|
|
|
|
|
|
|
15 | B | 58 | 44 | 53 | 52 ± 6 | 146 | 131 | 126 | 134 ± 8 | 77 | 22 | 10 |
Positive Control - MMS (µg/mL) | ||||||||||||
10 | -- | 169 | 182 | 176 | 176 ± 5 | 81 | 100 | 94 | 92 ± 8 | 383 | 328 | 37 |
20 | -- | 146 | 110 | 160 | 139 ± 21 | 27 | 31 | 33 | 30 ± 2 | 914 | 859 | 8 |
Rep = Replicate Solvent = DMSO ++ = Too toxic to clone * = Precipitating concentration aMutant frequency (per 10⁶ surviving cells) = (Average # TFT colonies / average # VC colonies) x 200 bInduced mutant frequency (per 10⁶ surviving cells) = mutant frequency - average mutant frequency of solvent controls c% Total growth = (% suspension growth x % cloning growth) / 100 | ||||||||||||
Cloning Data for L5178Y/TK+/- Mouse Lymphoma Cells Treated with DMBPC in the Presence of Exogenous Metabolic Activation Initial Assay (4-hour exposure)
Dose Level (µg/mL) | Rep | TFT Colonies | VC Colonies | Mutant Freq.a | Induced Mutant Freq.b | % Total Growthc | ||||||
Counts | Mean | Counts | Mean | |||||||||
0 (solvent) | 1 | 47 | 44 | 19 | 37 ± 13 | 148 | 155 | 177 | 160 ± 12 | 46 | -- | -- |
0 (solvent) | 2 | 26 | 53 | 40 | 40 ± 11 | 158 | 198 | 151 | 169 ± 21 | 47 | -- | -- |
Mean Solvent Mutant Frequency = 46 | ||||||||||||
15 | A | 37 | 37 | 26 | 33 ± 5 | 140 | 146 | 122 | 136 ± 10 | 49 | 3 | 90 |
15 | B | 19 | 19 | 42 | 27 ± 11 | 133 | 160 | 156 | 150 ± 12 | 36 | -11 | 101 |
20 | A | 33 | 48 | 39 | 40 ± 6 | 157 | 136 | 146 | 146 ± 9 | 55 | 8 | 93 |
20 | B | 19 | 17 | 35 | 24 ± 8 | 126 | 155 | 135 | 139 ± 12 | 34 | -12 | 93 |
25 | A | 37 | 34 | 37 | 36 ± 1 | 127 | 133 | 140 | 133 ± 5 | 54 | 8 | 68 |
25 | B | 18 | 15 | 35 | 23 ± 9 | 143 | 103 | 106 | 117 ± 18 | 39 | -8 | 67 |
30 | A | 34 | 32 | 50 | 39 ± 8 | 123 | 113 | 133 | 123 ± 8 | 63 | 16 | 30 |
30 | B | 28 | 20 | 31 | 26 ± 5 | 138 | 144 | 132 | 138 ± 5 | 38 | -8 | 58 |
35 | A | 32 | 24 | 17 | 24 ± 6 | 151 | 156 | 148 | 152 ± 3 | 32 | -14 | 16 |
35 | B | 33 | 26 | 51 | 37 ± 11 | 135 | 149 | 124 | 136 ± 10 | 54 | 8 | 15 |
Positive Control – 7,12-DMBA (µg/mL) | ||||||||||||
1 | -- | 81 | 90 | 85 | 85 ± 4 | 139 | 135 | 114 | 129 ± 11 | 132 | 86 | 68 |
1.5 | -- | 160 | 173 | 158 | 164 ± 7 | 103 | 111 | 116 | 110 ± 5 | 298 | 251 | 46 |
Rep = Replicate Solvent = DMSO aMutant frequency (per 10⁶ surviving cells) = (Average # TFT colonies / average # VC colonies) x 200 bInduced mutant frequency (per 10⁶ surviving cells) = mutant frequency - average mutant frequency of solvent controls c% Total growth = (% suspension growth x % cloning growth) / 100 | ||||||||||||
Summary of Test Results
Treatment (µg/mL) | S9 Activation | Treatment Time (hours) | Mean Mitotic Index | Cells Scored | Aberrations Per Cell (Mean ± SD) | Cells with Numerical Aberrations (%) | Cells with Structural Aberrations (%) |
Vehicle (DMSO) | - | 4 | 9.0 | 200 | 0.035 ± 0.184 | 1.5 | 3.5 |
DMBPC | |||||||
2.5 | - | 4 | 8.9 | 200 | 0.055 ± 0.250 | 1.5 | 5.0 |
10 | - | 4 | 9.8 | 200 | 0.065 ± 0.267 | 1.5 | 6.0 |
25 | - | 4 | 8.1 | 200 | 0.065 ± 0.247 | 1.0 | 6.5 |
Positive control (MMC) 0.2 | - | 4 | 9.4 | 200 | 0.190 ± 0.474 | 2.0 | 16.0** |
| |||||||
Vehicle (DMSO) | + | 4 | 10.6 | 200 | 0.010 ± 0.100 | 4.0 | 1.0 |
DMBPC | |||||||
2.5 | + | 4 | 11.0 | 200 | 0.055± 0.229 | 5.0 | 5.5* |
5 | + | 4 | 10.9 | 200 | 0.095±0.311 | 4.0 | 9.0** |
15 | + | 4 | 3.3 | 200 | 0.140± 0.770 | 1.0 | 8.5** |
Positive control (CP) 10 | + | 4 | 6.9 | 100 | 0.610± 0.886 | 4.0 | 40.0** |
| |||||||
Vehicle (DMSO) | - | 20 | 9.9 | 200 | 0.035 ± 0.184 | 1.5 | 3.5 |
DMBPC | |||||||
2.5 | - | 20 | 7.6 | 200 | 0.050 ± 0.218 | 2.0 | 5.0 |
10 | - | 20 | 8.7 | 200 | 0.060 ± 0.295 | 1.5 | 5.0 |
17.5 | - | 20 | 3.2 | 200 | 0.085 ± 0.297 | 1.5 | 8.0* |
Positive control (MMC) 0.1 | - | 20 | 6.9 | 200 | 0.255 ± 0.540 | 2.0 | 21.0** |
* p less than or equal to 0.05 (Fisher’s exact test) ** p less than or equal to 0.01 (Fisher’s exact test) | |||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Negative in male and female ICR mice (micronucleus assay); OECD 474
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- ICR
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- The ICR mice were obtained from the supplier and were approximately 6-8 weeks of age at study initiation, weighing 24.5 to 31.4 g (males) and 23.6 to 28.3 g (females). Up to five mice of the same sex were group housed in polycarbonate cages with heat-treated hardwood chips for bedding. The controlled environmental parameters were 72 ± 3 °F, 50 ± 20 % relative humidity and a 12-hour light/dark cycle. The mice had free access to certified rodent chow and tap water.
- Route of administration:
- intraperitoneal
- Vehicle:
- Corn oil (from ICN Biomedicals)
- Details on exposure:
- A workable suspension of the test substance in corn oil at 100 mg/mL – the maximum concentration tested in the study.
In the pilot study, DMBPC was administered to 2 male mice each at 1, 10, 100, or 1000 mg/kg and to 5 male and 5 and female mice at 2000 mg/kg. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration.
In the toxicity assay, 5 male and 5 female mice each were dosed with 200, 400, 600 or 800 mg test article/kg body weight. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration.
Mortality was observed in 5/5 male mice and 3/5 female mice at 400 mg/kg and in all males and females at 600 and 800 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in males and females at 200, 400, 600 and 800 mg/kg. In addition, ataxia was seen in females at 800 mg/kg. The high dose for the micronucleus test was set at 250 mg/kg, which was estimated to be the maximum tolerated dose.
For the definitive micronucleus assay, mice were assigned to seven groups, each containing 5 males and 5 females. Animals in five of these groups were treated either with the controls (negative or positive) or with DMBPC at a dose of 62.5, 125 or 250 mg/kg and were euthanised 24 hours after treatment. Animals in the other two groups were treated either with the negative control or DMBPC at a dose of 250 mg/kg and were euthanised 48 hours after treatment. Additional replacement animals (5 animals/sex) were included in the high dose group, 250 mg/kg, to ensure that the availability of 5 animals/sex for micronucleus analysis. DMBPC vehicle mixture, the vehicle alone or the positive control was administered by a single IP injection at a dose volume of 20 mL/kg body weight. All mice in the experimental and control groups were weighed immediately before dose administration and the dose volume was based on individual body weight. Mice were observed after dose administration for clinical signs of toxicity. - Frequency of treatment:
- Single treatment
- Post exposure period:
- 3 days
- Remarks:
- Doses / Concentrations: 1, 10, 100, 1000 or 2000 mg/kg body weight (nominal) - Pilot assay
- Remarks:
- Doses / Concentrations: 200, 400, 600 or 800 mg/kg body weight (nominal) - Toxicity assay
- Remarks:
- Doses / Concentrations: 62.5, 125 or 250 mg/kg body weight (nominal) - Definitive assay
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide monohydrate (CP, CAS No. 6055-19-2); 50 mg/kg
- Tissues and cell types examined:
- Bone marrow
- Details of tissue and slide preparation:
- At the scheduled sacrifice times, five mice per sex per dose were sacrificed by CO₂ asphyxiation. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May Gruenwald Giemsa and permanently mounted.
Bone marrow cells [polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)], were analysed for the presence of micronuclei. Polychromatic erythrocytes are young, immature red blood cells that stain bluish while normochromatic erythrocytes or normocytes are mature red blood cells that stain pink. Micronuclei are round, darkly-staining nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei can occur in both PCEs (MPCEs) and NCEs (MNCEs).
To control for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes (PCEs/ECs ratio). - Evaluation criteria:
- To quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and dose group.
As a guide to interpretation of the data, a test substance was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p less than or equal to 0.05, Kastenbaum Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time. - Statistics:
- The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and dose group. Statistical significance was determined using the Kastenbaum Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- In the pilot study, DMBPC was administered to two male mice each at 1, 10, 100, and 1000 mg/kg and to five male and five and female mice at 2000 mg/kg. Mortality was observed in 2/2 male mice at 1000 mg/kg and in 5/5 male mice and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in all male mice at 1, 10, 100 and 1000 mg/kg and in all male and female mice at 2000 mg/kg.
In the toxicity assay, five male and five female mice each were dosed with 200, 400, 600 or 800 mg test article/kg body weight. Mortality was observed in 5/5 male mice and 3/5 female mice at 400 mg/kg and in all males and females at 600 and 800 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in males and females at 400, 600 and 800 mg/kg. In addition, ataxia was seen in 4/5 females at 800 mg/kg. No clinical signs were observed in any of the animals in the 200 mg/kg dose group. The high dose for the micronucleus test was set at 250 mg/kg, which was estimated to be the maximum tolerated dose.
In the definitive micronucleus test, male and female mice were dosed with DMBPC by a single intraperitoneal injection of 62.5, 125 or 250 mg/kg as well as with the vehicle (corn oil) and positive control (CP) articles. The vehicle and dosing formulations were administered in a total volume of 20 mL/kg body weight. No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs following dose administration included: lethargy and piloerection in all male mice and all female mice at 250 mg/kg. All other animals treated with the test or control articles appeared normal following dose administration.
Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Slight to moderate reductions of 12 to 33 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test article to the bone marrow target. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time (p greater than 0.05, Kastenbaum-Bowman Tables). CP induced a statistically significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p less than or equal to 0.05, Kastenbaum-Bowman Tables).
In this study, all criteria for a valid test were met as specified in the protocol. CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p less than or equal to 0.05, Kastenbaum-Bowman Tables). The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or the quality of the test. - Conclusions:
- Under the conditions of this study, DMBPC was concluded to be negative in the micronucleus test using male and female ICR mice.
- Executive summary:
The potential of the test material to cause in vivo genotoxicity was investigated in a micronucleus assay conducted in accordance with the standardised guideline OECD 474 under GLP conditions.
Male and female ICR mice were exposed to the test material via the intraperitoneal route in corn oil. Following a pilot study and toxicity assay, for the definitive micronucleus assay mice were assigned to seven groups, each containing 5 males and 5 females. Animals in five of these groups were treated either with the controls (negative or positive) or with DMBPC at a dose of 62.5, 125 or 250 mg/kg and were euthanised 24 hours after treatment. Animals in the other two groups were treated either with the negative control or DMBPC at a dose of 250 mg/kg and were euthanised 48 hours after treatment. Mice were observed after dose administration for clinical signs of toxicity.
Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow collected and processed. Two slides for examination were prepared from each mouse. Bone marrow cells [polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)], were analysed for the presence of micronuclei; 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes (PCEs/ECs ratio).
In the definitive micronucleus test, no mortality occurred at any dose level during the course of the micronucleus study. Clinical signs following dose administration included: lethargy and piloerection in all male mice and all female mice at 250 mg/kg. All other animals treated with the test or control articles appeared normal following dose administration.
Slight to moderate reductions of 12 to 33 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test article to the bone marrow target. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time. CP induced a statistically significant increase in micronucleated polychromatic erythrocytes in both male and female mice. All criteria for a valid test were met.
Under the conditions of this study, a single intraperitoneal administration of DMBPC at doses up to 250 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, DMBPC was concluded to be negative in the micronucleus test using male and female ICR mice.
Reference
Summary of Bone Marrow Micronucleus Analysis Following a Single Dose of DMBPC (CAS No.2362-14-3) in ICR Mice
Treatment | Sex | Time (hr) | Number of Mice | PCE/Total Erythrocytes (Mean ± SD) | Change from Control (%) | Micronucleated Polychromatic Erythrocytes | |
Number per 1000 PCEs (Mean ± SD) | Number per PCEs Scored | ||||||
Corn oil | M | 24 | 5 | 0.469 ± 0.09 | --- | 0.4 ± 0.42 | 4 / 10000 |
F | 24 | 5 | 0.390 ± 0.09 | --- | 0.4 ± 0.42 | 4 / 10000 | |
DMBPC | |||||||
62.5 mg/kg | M | 24 | 5 | 0.409 ± 0.04 | -13 | 0.3 ± 0.27 | 3 / 10000 |
F | 24 | 5 | 0.397 ± 0.08 | 2 | 0.4 ± 0.22 | 4 / 10000 | |
125 mg/kg | M | 24 | 5 | 0.347 ± 0.02 | -26 | 0.4 ± 0.22 | 4 / 10000 |
F | 24 | 5 | 0.344 ± 0.07 | -12 | 0.5 ± 0.35 | 5 / 10000 | |
250 mg/kg | M | 24 | 5 | 0.312 ± 0.03 | -33 | 0.4 ± 0.22 | 4 / 10000 |
F | 24 | 5 | 0.321 ± 0.07 | -18 | 0.5 ± 0.00 | 5 / 10000 | |
CP | |||||||
50 mg/kg | M | 24 | 5 | 0.317 ± 0.02 | -32 | 26.0 ± 3.02 | *260 / 10000 |
F | 24 | 5 | 0.289 ± 0.04 | -26 | 27.4 ± 4.72 | *274 / 10000 | |
Corn oil | M | 48 | 5 | 0.415 ± 0.05 | --- | 0.1 ± 0.22 | 1 / 10000 |
F | 48 | 5 | 0.392 ± 0.04 | --- | 0.5 ± 0.00 | 5 / 10000 | |
DMBPC | |||||||
250 mg/kg | M | 48 | 5 | 0.351 ± 0.09 | -15 | 0.3 ± 0.27 | 3 / 10000 |
F | 48 | 5 | 0.327 ± 0.01 | -17 | 0.2 ± 0.27 | 2 / 10000 | |
*Statistically significant, p less than or equal to 0.05 (Kastenbaum‑Bowman Tables) | |||||||
Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 24 Hours Following a Single Dose of DMBPC (CAS No. 2362 -14 -3)in ICR Mice
Treatment (mg/kg) | Sex | Animal Number | PCE/Total Erythrocytes | Micronucleated PCE (Number/PCE scored) |
Corn oil (20 mL/kg) | M | 101 | 0.323 | 1 / 2000 |
102 | 0.455 | 2 / 2000 | ||
103 | 0.507 | 1 / 2000 | ||
104 | 0.548 | 0 / 2000 | ||
105 | 0.513 | 0 / 2000 | ||
F | 106 | 0.346 | 0 / 2000 | |
107 | 0.436 | 1 / 2000 | ||
108 | 0.325 | 2 / 2000 | ||
109 | 0.528 | 0 / 2000 | ||
110 | 0.314 | 1 / 2000 | ||
DMBPC | ||||
62.5 | M | 111 | 0.413 | 0 / 2000 |
112 | 0.351 | 1 / 2000 | ||
113 | 0.454 | 0 / 2000 | ||
114 | 0.421 | 1 / 2000 | ||
115 | 0.408 | 1 / 2000 | ||
F | 116 | 0.467 | 1 / 2000 | |
117 | 0.420 | 1 / 2000 | ||
118 | 0.266 | 0 / 2000 | ||
119 | 0.410 | 1 / 2000 | ||
120 | 0.423 | 1 / 2000 | ||
125 | M | 121 | 0.371 | 1 / 2000 |
122 | 0.326 | 0 / 2000 | ||
123 | 0.364 | 1 / 2000 | ||
124 | 0.317 | 1 / 2000 | ||
125 | 0.358 | 1 / 2000 | ||
F | 126 | 0.324 | 1 / 2000 | |
127 | 0.276 | 1 / 2000 | ||
128 | 0.466 | 0 / 2000 | ||
129 | 0.312 | 2 / 2000 | ||
130 | 0.341 | 1 / 2000 | ||
250 | M | 131 | 0.284 | 1 / 2000 |
132 | 0.361 | 1 / 2000 | ||
133 | 0.323 | 0 / 2000 | ||
134 | 0.277 | 1 / 2000 | ||
135 | 0.315 | 1 / 2000 | ||
F | 136 | 0.341 | 1 / 2000 | |
137 | 0.233 | 1 / 2000 | ||
138 | 0.416 | 1 / 2000 | ||
139 | 0.325 | 1 / 2000 | ||
140 | 0.288 | 1 / 2000 | ||
CP 50 | M | 141 | 0.340 | 48 / 2000 |
142 | 0.328 | 50 / 2000 | ||
143 | 0.324 | 55 / 2000 | ||
144 | 0.317 | 61 / 2000 | ||
145 | 0.277 | 46 / 2000 | ||
F | 146 | 0.230 | 48 / 2000 | |
147 | 0.269 | 46 / 2000 | ||
148 | 0.300 | 70 / 2000 | ||
149 | 0.321 | 54 / 2000 | ||
150 | 0.324 | 56 / 2000 | ||
Induction of Micronucleated Polychromatic Erythrocytes inBone Marrow Cells Collected 48 Hours Following a Single Dose of DMBPC
(CAS No. 2362 -14 -3) in ICR Mice
Treatment (mg/kg) | Sex | Animal Number | PCE/Total Erythrocytes | Micronucleated PCE (Number/PCE scored) |
Corn oil | M | 151 | 0.405 | 0 / 2000 |
152 | 0.430 | 0 / 2000 | ||
153 | 0.488 | 0 / 2000 | ||
154 | 0.415 | 0 / 2000 | ||
155 | 0.336 | 1 / 2000 | ||
F | 156 | 0.408 | 1 / 2000 | |
157 | 0.328 | 1 / 2000 | ||
158 | 0.374 | 1 / 2000 | ||
159 | 0.413 | 1 / 2000 | ||
160 | 0.438 | 1 / 2000 | ||
DMBPC | ||||
250 | M | 161 | 0.473 | 1 / 2000 |
162 | 0.312 | 1 / 2000 | ||
163 | 0.272 | 0 / 2000 | ||
164 | 0.288 | 1 / 2000 | ||
165 | 0.411 | 0 / 2000 | ||
F | 166 | 0.342 | 1 / 2000 | |
167 | 0.320 | 1 / 2000 | ||
168 | 0.328 | 0 / 2000 | ||
169 | 0.333 | 0 / 2000 | ||
170 | 0.314 | 0 / 2000 | ||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro bacterial reverse mutation assay
The potential of the test material to cause genetic toxicity was investigated in a bacterial reverse mutation assay conducted in accordance with the standardised guideline OECD 471 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA were exposed to the test material in DMSO via the plate incorporation method at dose levels of 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 micrograms per plate with and without S9 activation in a preliminary toxicity and initial mutagenicity assay. The strains were then exposed to 5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate in an independent repeat/confirmatory mutagenicity assay.
Under the conditions of this study, the test material was negative in both the presence and absence of metabolic activation.
In vitro mammalian cell gene mutation assay
The genotoxicity potential of the test material was investigated in a mammalian cell gene mutation assay conducted in accordance with the standardised guideline OECD 476 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Mouse lymphoma L5178Y TK+/- cells were exposed to the test material in DMSO at the following concentrations: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 3000 µg/mL with and without S9 activation in a preliminary assay; 5, 7.5, 10, 12.5, 15 and 17.5 µg/mL in the 4 hour mutagenicity assay without S9 activation; and 15, 20, 25, 30 35 and 40 µg/mL in the 4 hour mutagenicity assay with S9 activation.
Under the conditions of this study, the test material was considered to be negative with and without metabolic activation in the L5178Y TK+/- mouse lymphoma mutagenesis assay.
In vitro mammalian chromosome aberration test
The genotoxicity potential of the test material was investigated in an in vitro mammalian chromosome aberration test conducted in accordance with the standardised guideline OECD 473 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
Chinese hamster ovary cells (CHO-K1) were exposed to the test material in DMSO at the following concentrations: 0.296, 0.888, 2.96, 8.88, 29.6, 88.8, 296, 888 and 2960 µg/mL in the preliminary toxicity assay; 2.5, 5, 10, 12.5, 15, 17.5, 20, and 25 µg/mL in the 4 hour chromosome aberration assay without S9 activation; and 1.25, 2.5, 5, 10, 12.5, 15, 17.5, 20, and 25 µg/mL in the 4 hour chromosome aberration assay with S9 activation and 20-hour assay without S9 activation.
Toxicity of DMBPC in CHO cells treated for 4-hours without metabolic (S9) activation was 61 % at 25 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest test concentration evaluated for chromosome aberrations (25 µg/mL) was 10 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 10 and 25 µg/mL. The percentage of cells with structural and numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control.
Toxicity of DMBPC in CHO cells when treated for 4-hours with S9 activation was 55 % at 15 µg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest test concentration evaluated for chromosome aberrations (15 µg/mL) was 69 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 5.0 and 15 µg/mL. The percentage of cells with structural aberrations in the test article-treated group was statistically increased above that of the solvent control at all concentrations evaluated. However, the percentage of cells with structural aberrations (5.5 %) in the DMBPC-treated groups at 2.5 µg/mL was within the historic solvent control range of 0.0 to 6.5 %. Therefore, this would not be considered biologically significant. The percentage of structurally aberrant cells at concentrations of 5 and 15 µg/mL, 9.0 and 8.5 %, respectively, fall just outside the historical solvent control range and this is considered to be positive. The percentage of cells with numerical aberrations in the test article-treated groups was not significantly increased above that of the solvent control.
In the absence of a positive response in the non-activated 4 hour exposure group, slides from the non-activated 20-hour exposure group were evaluated for chromosome aberrations. Toxicity of DMBPC in CHO cells was 31 % at 17.5 µg/mL, the highest test concentration evaluated for chromosome aberrations in the non-activated 20-hour continuous exposure. The mitotic index at the highest test concentration evaluated for chromosome aberrations (17.5 µg/mL) was 68 % reduced relative to the solvent control. The dose levels selected for microscopic analysis were 2.5, 10 and 17.5 µg/mL. The percentage of cells with structural aberrations in the test article-treated groups was significantly increased above that of the solvent control at 17.5 µg/mL. The percentage of cells with structural aberrations in the DMBPC-treated at 17.5 µg/mL group (8.0 %) was outside the historical solvent control range of 0.0 to 6.0 % and would be considered positive for the induction of structural chromosome aberrations. The percentage of cells with numerical aberrations in the DMBPC-treated groups was not statistically significantly increased above that of the solvent controls at any dose level.
Based on the findings of this study, DMBPC was concluded to be positive for the induction of structural chromosome aberrations in CHO cells in the S9 activated 4-hour exposure group and in the non-activated 20-hour exposure group. DMBPC was concluded to be negative for the induction of numerical chromosome aberrations in CHO cells.
In vivo micronucleus assay
The potential of the test material to cause in vivo genotoxicity was investigated in a micronucleus assay conducted in accordance with the standardised guideline OECD 474 under GLP conditions.
Male and female ICR mice were exposed to the test material via the intraperitoneal route in corn oil. Following a pilot study and toxicity assay, for the definitive micronucleus assay mice were assigned to seven groups, each containing 5 males and 5 females. Animals in five of these groups were treated either with the controls (negative or positive) or with DMBPC at a dose of 62.5, 125 or 250 mg/kg and were euthanised 24 hours after treatment. Animals in the other two groups were treated either with the negative control or DMBPC at a dose of 250 mg/kg and were euthanised 48 hours after treatment. Mice were observed after dose administration for clinical signs of toxicity.
Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow collected and processed. Two slides for examination were prepared from each mouse.
Under the conditions of this study, a single intraperitoneal administration of DMBPC at doses up to 250 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, DMBPC was concluded to be negative in the micronucleus test using male and female ICR mice.
Discussion
DMBPC was negative for mutagenicity in the in vitro bacterial reverse mutation assay (BioReliance, 2001a), as well as the in vitro L5178Y TK +/- Mouse Lymphoma Forward Mutation Assay (BioReliance, 2001b). The overall weight-of-evidence demonstrates that DMBPC is not mutagenic.
In the in vitro chromosome aberration assay, DMBPC was weakly positive for induction of structural chromosome aberrations in CHO cells, but it was negative for numerical chromosome aberrations in CHO cells (BioReliance, 2001c). In the in vivo micronucleus assay, a single intraperitoneal administration of DMBPC at doses up to 250 mg/kg bw did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow (BioReliance, 2001d). Therefore, DMBPC was concluded to be negative in the in vivo micronucleus test.
The in vitro chromosomal aberration assay can detect large chromosomal damage and aneuploidy, while the in vivo micronucleus assay can detect small chromosomal damage, large chromosomal damage, and aneuploidy (GLP/OECD Guideline 473 and 474). Considering that the in vitro chromosomal aberration assay and the in vivo micronucleus assay detect similar types of chromosomal damage, and that the weakly positive results in the in vitro chromosome aberration assay were not confirmed by the negative results of the in vivo micronucleus test, coupled with the fact that in vivo studies are considered to be the most biologically relevant, the weight-of-evidence demonstrates that DMBPC is not mutagenic.
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.
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