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Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Protocols and results reviewed and accepted by the National Toxicology Program's Board of Scientific Counselor's Technical Reports Review Subcommittee, USA National Institutes of Health
Justification for type of information:
Please see Category Approach
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine independent mutant colonies of Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 and hamster S9
Test concentrations with justification for top dose:
100, 333, 1000, 3333 and 10000 microgram active ingredient/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: buffer
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, sodium azide, 4-niro-o-phenylenediamine, nad 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period: no data- Exposure duration: 2 days- Expression time (cells in growth medium): - Selection time (if incubation with a selection agent): - Fixation time (start of exposure up to fixation or harvest of cells): SELECTION AGENT (mutation assays): SPINDLE INHIBITOR (cytogenetic assays): STAIN (for cytogenetic assays): NUMBER OF REPLICATIONS: triplicates and entire trial repeated onceNUMBER OF CELLS EVALUATED: histidine independent coloniesDETERMINATION OF CYTOTOXICITY - Method: mitotic index; cloning efficiency; relative total growth; other:OTHER EXAMINATIONS: - Determination of polyploidy: - Determination of endoreplication: - Other: OTHER:
Evaluation criteria:
positive response defined as reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in revertants that is not dose-related, is not reproducible, or is of insufficient magnitude to support a determination of mutagenicity.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negativeTest substance is not mutagenic
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP compliance, Full documentation
Justification for type of information:
Please See Category Approach
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: TA98 and TA100 strains contain the pKM101 plasmid (carrying the R-factor)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
100, 330, 1000, 3333 and 5000 micrograms/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water- Justification for choice of solvent/vehicle:the relatively high solubility of the test substance
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
other: vehicle was water
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-nitrofluorene, sodium azide, 9-aminoacridine
Remarks:
positive controls varied with test strain
Details on test system and experimental conditions:
On the day of use, all tester strain cultures were checked for the correct genotype. The presence of the rfa wall mutation was confirmed by demonstration of sensitivity to crystal violet. The deletion in the uvrB gene was confirmed by demonstration of sensitivity to ultraviolet light. The presence of the pKM101 plasmid was confirmed by demonstration of resistance to ampicillin. Spontaneous reversion frequencies in the vehicle controls were demonstrated by plating 100 microliter aliquots of the culture along with the appropriate vehicle on selective media. To determine the sterility of the test substance, the highest dose (5000 microliters/plate) was plated on selective agar with an aliquot volume equal to that used in the assay. To determine the sterility of the S9 or Sham mix, a 0.5 mL aliquot of S9 or Sham mix was plated on selective agar.Test substance dilutions wer prepared immediately before use. 0.5 mL of S9 or Sham mix, 100 microliter of tester strain and 50 microliter of vehicle or test substance were added to 2 mL of agar at 45 degrees C. The positive control was substituted for the test substance. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. The plates were inverted and incubated for 48-72 hours at 37 +/- 2 degrees C. Plates were read immediately or else were stored at 4 degrees C until read.The dosing solutions reflected the active ingredient in the test substance. Condition of the bacterial background lawm was evaluated for evidence of test substance toxicity using a dissecting microscope.. Toxicity and precipitate were scored relative to the vehicle control plate. Revertant colonites were counted either entirely by automated colongy counter or entirely by hand (the later in the case of excess precipitate) All dose levels of test substance, vehicle controls and positive controls were plated in triplicate. For each replicate plating, the mean and standard deviation of the number of revertants per plate was calculated.
Evaluation criteria:
Validity criteria include: the presece of the deep rough mutation (rfa), the deletion in hte uvrB gene and the characteristic mean number of spontaneous revertants in the vehicle control of 10-50 for TA98, 80-240 for TA100, 5-45 for TA1535, 3-21 for TA1537, and 5-35 for TA1538. In addition, TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. Strain culture titers must be greater than or equal to 0.3 x 10-9 cells/mL. The mean of each positive control must be at least a 3X increase in revertants compared with respective vehicle control. A minimum of three non-toxic dose levels are required. A dose level is considered toxic if >50% reduction in revertants versus its control and/or a reduction in the background lawn. For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one strain with a minimum of two increasing concentrations of the test substance. 2X increases are needed for TA98 and TA100. 3X increases are needed for TA1535, TA1537 and TA 1538.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Historical control data are provided in Appendix IA range finding study was conducted to a maximum dose of 5000 micrograms active ingredient per plate. Neither precipitate nor appreciable toxicity was observed.In the definitive assay, neither precipitate nor appreciable toxicity was observed. No positive responses were observed with any of the strains in the presence and absence of S9 activation. All criteria for a valid study were met. The data are presented in Tables 2-11 and summarized in Table 12
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negativeThe test substance, SAR 33-55, is not genotoxic based on the results of the Ames Test
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Protocols and results reviewed and accepted by the National Toxicology Program's Board of Scientific Counselor's Technical Reports Review Subcommittee, USA National Institutes of Health
Justification for type of information:
Please see Category Approach
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
not specified
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 male Sprague-Dawley rat liver S9
Test concentrations with justification for top dose:
2513, 3750 and 5000 micrograms active ingredient per milliliter
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: with S9 and mitomycin-C without S9
Details on test system and experimental conditions:
Without S9, cells were incubated in McCoy's 5A medium with the test substance for 18 hours. colcemid was then added and incubation continued for additional 2 hours. The cells were harvested by mitotic shake-off, fixed and stained with Giemsa. The cells with S9 activation were treated with the test substance for 2 hours at which time the treatment medium was removed and the cells were incubated for 10 hours in fresh medium with Colcemid present for the last 2 hours. Harvesting was the same.
Evaluation criteria:
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 +/- 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each test level. Aberrations were classified as "simple" (breaks and terminal deletions), "complex" (rearrangements and translocations) and "other" (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).
Statistics:
Analyses were conducted on both the dose response curve and individual dose points. For a single trial, a significant (P
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negative with and without metabolic activationTest substance was not genotoxic in this assay
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Protocols and results reviewed and accepted by the National Toxicology Program's Board of Scientific Counselor's Technical Reports Review Subcommittee, USA National Institutes of Health
Justification for type of information:
Please see Category Approach
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
mouse lymphoma cells - TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
125, 250, 500, 1000 and 2500 microgram active ingredient per milliliter
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: without S9 and methlcholanthrene with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in Fischer mediumDURATIONNormal cycling time for L5178Y cells was 10 hours. Treated cultures contained 6 x 10-6 cells in 10mL medium. Incubation with test substance continued for 4 hours, at which time the medium plus test substance was removed and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was maintained at log phase growth. After the 48-hr expression period, cells were plated in medium and soft agar supplemented with TFT (trifluorothymidine) for selection of TFT-resistant cells. Cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 degrees C in 5% CO2 for 10-12 days. NUMBER OF REPLICATIONS: each treatment level was replicated, including positive and solvent controlsOTHER:
Evaluation criteria:
Minimum criteria for accepting an experiment as valid are presented in Caspary et al 1988
Statistics:
All data were evaluated statistically for trend and peak responses. Both responses had to be significant (P<=0.05) for the test substance to be considered positive; i.e., capable of inducing TFT resistance. A single significant response led to a "questionable" conclusion, and absence of both a trend and a peak response resulted in a "negative" conclusion.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Negative in one replicate series and positive in other replicate series with metabolic activation; leads to "equivocal" conclusion with metabolic activation
Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negative without metabolic activation questionable with metabolic activationNot mutagenic without metabolic activation, equivocal with metabolic activation
Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Protocols and results reviewed and accepted by the National Toxicology Program's Board of Scientific Counselor's Technical Reports Review Subcommittee, USA National Institutes of Health
Justification for type of information:
Please see Category Approach
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5900 - In vitro Sister Chromatid Exchange Assay
Deviations:
not specified
GLP compliance:
yes
Type of assay:
sister chromatid exchange assay in mammalian cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver S9
Test concentrations with justification for top dose:
500, 1667, 2513, 3750 and 5000 micrograms active substance per milliliter
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: with S9 and mitomycin-C without S9
Details on test system and experimental conditions:
Testing was performed as reported by Galloway et al 1987. In the test without S9, CHO cells were incubated for 25.5 hours with the test substance in McCoyy's supplemented 5A medium. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 25.5 hours, fresh medium was provided to include BrdU and Colcemid and further incubated for 2 hours. Cells were harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa.In the SCE test with S9, cells were incubated with the test substance, serum-free medium and S9 for 2 hours. Then the medium was replaced with medium containing serum and BrdU and no test substance and incubated for 25.5 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as cells treated without S9.
Evaluation criteria:
All slides were scored blind and those from a single test were read by the same person. fifty second-division metaphase cells were scored for frequency of SCEs/cell from each dose level.
Statistics:
Conducted on the slopes of the dose-response curves and the individual data points. An SCE frequency 20% above the concurrent solent control was chosen as a statistically conservative positive response. The probability of this level of difference occurring by chance at one dose is less than 0.01 and at two doses is less than 0.001. An increase of 20% or greater at any single dose was considered weak evidence of activity; increases at two or more doses was considered a positive response. A statistically significant trend in the absence of any responses reaching 20% above background was considered equivocal.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
with metabolic activation
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Clastogenic without metabolic activation at doses >1667 ug/mL. Not clastogenic with metabolic activation up to 5000 ug/mL. Cell cycle delay was apparent at concentrations >2513 ug/mL; incubation time was increased.
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negative with metabolic activation positive without metabolic activationTest substance not genotoxic with metabolic activation
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June to July 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented study according to OECD test guideline
Justification for type of information:
Please see Category Approach
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted May 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: BOR:NMRI (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Winkelmann, Borchen, Germany- Age at study initiation: young adults- Weight at study initiation: 30.4 +/- 1.7 g (males); 24.3 +/- 1.5 g (females)- Assigned to test groups randomly: no data- Fasting period before study: no- Housing: 5 animals per cage, gender separated, macrolone cages Type III- Diet : Ssniff R10 exclusive diet for rats (ad libitum); supplied by Ssniff Spezialfutter GmbH, Soest, Germany- Water: drinking water (ad libitum)- Acclimation period: one weekENVIRONMENTAL CONDITIONS- Temperature (°C): 20 +/- 3- Humidity (%): 30-70 - Air changes (per hr): no data- Photoperiod (hrs dark / hrs light): 12/12IN-LIFE DATES: From: July 9, 1991 To: July 18, 1991
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water- Justification for choice of solvent/vehicle: no data- Concentration of test material in vehicle: 40 %- Amount of vehicle: 16.8 ml/kg bw
Frequency of treatment:
single oral application
Post exposure period:
24, 48 and 72 hours
Remarks:
Doses / Concentrations:4467 mg/kg bwBasis:actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide- Justification for choice of positive control(s): no data- Route of administration: oral by gavage- Dose: 100 mg/kg bw- Vehicle: water- Total application volume: 10 ml/kg bw- post exposure period: 24 hours
Tissues and cell types examined:
bone marrow; polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The maximum tolerable dose (MTD)* was selected as dose.The MTD was determined in a dose range finding study :Phase 1: Limit test with 5000 mg/kg bwPhase 2: Determination of the TMTD range with reduced animal numberPhase 3: Determination of the MTD with 5 animals/sex/dose*MTD is defined as dose with no mortality but clear clinical symptoms within 3 days after applicationTREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):24, 48 and 72 hours after treatment the animals were killed by cervical dislocation and tissue was sampled.DETAILS OF SLIDE PREPARATION:The femora were removed and the bone marrow was suspended in fetal calf serum. The cell suspensions were centrifuged with 160 x g for 5 minutes and the supernatand discarded. The serum was resuspended and the suspension purified using a cellulose chromatographic column. The eluate was centrifuged at 800 x g for 10 minutes and the pellet in fetal calf serum /25 mM EDTA suspended. From this suspension 3-4 smears per animals were prepared on slides which were dried for at least 24 hours and stained with May-Grünwald/Giemsa solution.METHOD OF ANALYSIS: The cell analysis was performed by means of a Zeiss miscroscope at a 1000fold magnification (oil immersion). At least 1000 polychromatic erythrocytes (PCE) per animal were examined to determine the frequency of micronucleated cells. The ratio of PCE to normochromatic cells (NCO) was determined for a sample of 1000 erythrocytes. The number of micronucleated cells in counted NCE was determined.
Evaluation criteria:
For the identification of micronuclei the following criteria were considered:a) roundish and clear contour by the nuclear membraneb) diameter of about 1/20 of the size of the polychromatic erythrocytec) lays in the same focus layer as the observed erythrocyteThe micronucleus test is regarded as positive (test substance induces micronuclei in polychromatic erythrocytes) if the frequency of mucronucleated polychromatic erythrocytes of at least one tretament group is statistically significantly increased compared to the negative control and the increase is biologically relevant.
Statistics:
Mean values and standard deviations were calculated for the following parameters: a) number of polychromatic erythrocytes (PCE) containing micronucleib) ratio of PCE/NCEComparison of treatment groups with different post exposure periods with negative controls of respective post exposure periods. After control of the relative frequency of micronuclei in the treatment groups on homogeneity with the mean relative frequency a statistical analysis of micronucleus frequency using a 2 x 2 contingency table with chi² test and continuity table according to Yates was performed (see [1]).The differences of miconucleus frequencies in the positive control were reassessed in the two-sided t-test. This test was also used for the statistical analysis of the PCE/NCE-ratio.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY- Dose range: Phase 1: males and females: 5000 mg/kg bw (limit test); Phase 2: males: 1585, 1995, 2512, 3162 and 3981 mg/kg bw; females: 3162, 3548, 3981, 4467 and 5000 mg/kg bw. Phase 3: males and females: 3981, 4467 and 5000 mg/kg bw.- Clinical signs of toxicity in test animals: Phase 1: 4/5 male and 1/5 female animals died. The males died within 4 hours after application, the female died within 24 hours after application of the test substance. Clinical signs as hunched posture, ruffled fur, closed eyes and diarrhea were observed. Phase 2: 1/5 female at 3981 mg/kg bw died. At high doses clinicals signs as hunched posture, ruffled fur, closed eyes and diarrhea were observed. Phase 3: 2/5 male and 2/5 female animals died at 500 mg/kg bw. At 4467 mg/kg bw no mortality occured but severe toxic effects.RESULTS OF DEFINITIVE STUDY- Clinical signs of toxicity in test animals: Hunched posture, ruffled fur, closed eyes and diarrhea were observed. One day after application the effects subsided and two days after application all animals were free of clinical symptoms. One male and one female animal died 24 and 5.5 hours after application, respectively. These animals were replaced with mice of a concurrent satellite group also treated with the test substance. One male of the satellite group died 5 hours after application.- Induction of micronuclei: Positive control: Significantly increased number of polychromatic erythrocytes (PCE) with miconuclei both in male and female animals. Test substance: No significant increase in the number of PCE with micronuclei after 24 and 48 hours in both males and females and after 72 hours in males. After 72 hours a statistically significant increase of the number of micronuclei in PCEs in females were observed. If the results of males and females were combined no significant difference in the frequency of micronuclei between treatment group and combined vehicle control group is observed. (Tables 1, 2 and 3)- Ratio of PCE/NCE: Neither in the positive control group nor in the treated males a significant difference of PCE/NCE ratio compared to the control group is observed. In the females of the treatment groups no difference was observed 24 and 48 hours after treatment. After 72 hours the PCE/NCE ratio was decreased compared to vehicle control group but still was above the typically observed ratio of 1.0. (Tabels 1, 2 and 3)- Appropriateness of dose levels and route: The selected dose level investigated as the maximum tolerable dose (MTD) induced clear toxic symptoms and was lethal in two animals. According to the guideline the recommended criteria for the dose level were fulfilled. The route of administration was appropriate as systemic availability could be demonstrated by clinical signs.

The statistically significant increase in frequency of micronuclei is considered to be of no biological relevance for the following reasons:

- The frequency of micronuclei at this sampling time point (0.18%) is not above the frequency of micronuclei of controls generally observed in this test laboratory (0.07 - 0.22%). The statistical significance in this case is caused by the low frequency of micronuclei in the control group (0.02%) which deviates downwards from the so far observed frequencies for controls. After addition of male and female animals of sampling time point 72 h into one group there is no more a statistically significant difference.

- A delayed effect based on slow excretion is improbable for sodium cumenesulphonate as sulphonic acids in general are readily absorbed and do not show any tendency for accumulation. But this kind of detention is regarded as prerequisite to explain based on the kinetic of erythrocyte maturation an impact on micronuclei frequency at sampling time point of 72 hours.

The results of the positive control affirm the sensitivity of the mouse strain to mutagenic substances.The frequency of micronuclei in polychromatic erythrocytes was considerably increased compared to the control group.

Table 1: Results of in vivo micronucleus test for male animals (mean ± standard deviation)

      Neg. control   test substance 4467 mg/kg bw      Pos. control
  sampling time    24 h  48 h  72 h  24 h  48 h  72 h 24 h 
micronuclei in 1000 PCE  0.8  ± 0.8 1.8  ± 0.8   0.4 ± 0.5 0.6 ± 0.5    0.8  ± 0,8   0.2  ± 0.4 48.0* ± 19.6
 % PCE with micronuclei 0.08  0.18  0.04 0.06  0.08 0.02  4.8
PCE / NCE  1.0 ± 0.2 1.4 ± 0.3  1.6 ± 0.3  1.0 ± 0.3 1.1 ± 0.4   1.9 ± 0.9 0.8  ± 0.1

*p < 0.05

Table 2: Results of in vivo micronucleus test for female animal (mean ± standard deviation)

      Neg. control   test substance 4467 mg/kg bw      Pos. control
  sampling time    24 h  48 h  72 h  24 h  48 h  72 h 24 h 
micronuclei in 1000 PCE 1.4  ± 1.7 1.8  ± 1.1   0.2 ± 0.4 2.2 ± 1.1    1.2  ± 0,8   1.8*  ± 1.5 36.8* ± 9.8
 % PCE with micronuclei 0.14  0.18  0.02 0.22  0.12 0.18 3.6
PCE / NCE  1.2 ± 0.2 1.4 ± 0.2  2.4 ± 0.6  1.0 ± 0.2 1.3 ± 0.3   1.3 ± 0.5 1.0  ± 0.1

*p< 0.05

Table 3: Results of in vivo micronucleus test for male + female animals (mean ± standard deviation)

      Neg. control   test substance 4467 mg/kg bw      Pos. control
  sampling time    24 h  48 h  72 h  24 h  48 h  72 h 24 h 
micronuclei in 1000 PCE  1.1  ± 1.3 1.8  ± 0.9  0.3 ± 0.5 1.4 ± 1.2    1.0  ± 0.8   1.0  ± 1.3 42.4* ± 15.7
 % PCE with micronuclei 0.11  0.18  0.03 0.14  0.10 0.10  4.24
PCE / NCE  1.1 ± 0.2 1.4 ± 0.3  2.0 ± 0.6  1.0 ± 0.2 1.2 ± 0.4   1.6 ± 0.8 0.9  ± 0.1

* p < 0.05

Conclusions:
Interpretation of results (migrated information): negativeAll male mice treated with the test substance showed no statistically significant increase in micronucleus frequency at any sampling time compared to control animals. For the female mice treated with the test substance at sampling times 24 and 48 hours after treatment also no statistically significant increase in micronucleus frequency was observed. Only at sampling time point 72 hours a statistically significant increase of polychromatic erythrocytes with micronuclei compared to control animals was observed. This effect was regarded as biologically not relevant as this increase ís based on the exceptional low micronucleus frequency of vehicle control group.Sodium cumenesulphonate under these test conditions is regarded as not mutagenic in the micronucleus test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

There are 5 different in vitro genetic toxicity assays and 2 different in vivo genetic toxicity assays conducted with hydrotrope substances, principally sodium xylene sulphonate. The key studies are as follows:

 

An in vitro bacterial cell gene mutation study (Ames Test) on sodium xylene sulphonate with four strains of Salmonella, all with and without S9 metabolic activation, positive and negative controls, and 5 exposure concentrations of the test substance. The genotoxicity results were negative for all conditions.

An in vitro mammalian cell gene mutation study (Mouse Lymphoma) on sodium xylene sulphonate with and without S9 metabolic activation, positive and negative controls, a DMSO vehicle, and 5 exposure concentrations of the test substance. The genotoxicity results were negative without activation and equivocal with activation (one replicate was positive and the 2nd replicate was negative). The study conclusion was "negative genotoxicity".

A mammalian cell DNA damage assay (Sister Chromatid Exchange in Chinese Hamster Ovary) on sodium xylene sulphonate with and without Aroclor 1254 activation, positive and negative controls, using a DMSO vehicle, and 5 exposure concentrations of the test substance. The genotoxicity results were negative with activation and positive without activation (clastogenic at doses of 2513 micrograms active per milliliter and higher). The study conclusion was not genotoxic with metabolic activation.

A mammalian cell chromosome aberration study on sodium xylene sulphonate in Chinese Hamster Ovary cells with and without Aroclor 1254 metabolic activation, positive and negative control, tested up to limit concentrations. The test material was not genotoxic in this assay.

An in vivo micronucleus study on sodium cumene sulphonate where mice were dosed by oral gavage at 4467 mg/kg bw. The study gave a negative result.

 

The other supporting studies also gave negative results.


Short description of key information:
There are 5 different in vitro genetic toxicity assays and 2 different in vivo genetic toxicity assays conducted with hydrotrope category substances, principally sodium xylene sulphonate. The assays are Ames Test, mouse lymphoma mammalian gene mutation, Chinese hamster sister chromatid exchange, Chinese hamster ovary mammalian chromosome aberration, in vivo mouse micronucleus bone marrow chromosome analysis, and in vivo mammalian erythrocyte micronucleus. All are guideline studies, conducted according to GLP requirements and fully documented. The conclusion from the battery of 7 different validated assays is the hydrotrope substances are not mutagenic or genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

On the basis of the negative results obtained across a range of in vitro and in vivo assays, there is no justification for classification.