Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro, OECD 471, CAS 70024-69-0, negative
Chromosome aberration, OECD 473, CAS 71786-47-5, negative

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study has been conducted to OECD guidelines and to GLP, and therefore meets the criteria for Klimisch code 1. However as this study is used in a read-across approach Klimisch 2 is assigned.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
araclor
Test concentrations with justification for top dose:
0.1, 0.33, 1.0, 3.33 and 10 mg/plate
Vehicle / solvent:
pluronic F127 25 % w/w in ethanol
Negative solvent / vehicle controls:
yes
Remarks:
Pluronic F127 25% w/w in ethanol
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: 2-aminoanthracene and ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3/dose group/strain/treatment set

Evaluation criteria:
Number of revertant colonies.
Statistics:
Mean revertant colony count and standard deviation were determined for each dose point.
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
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No reduction in the number of revertants/plate was observed in the range-finding study with strains TA100 and WP2uvrA.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in the dose range-finding study with strains TA100 and WP2uvrA with or without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

No cytotoxic response was seen in the dose range finding study.

The positive control exhibited at least a 3 fold increase in revertant colonies.

Results: The test substance was not genotoxic in this assay with or without metabolic activation.

Remarks

No cytotoxicity was observed in the dose range-finding study with tester strains TA100 and WP2uvrA with or without metabolic activation as evidenced by normal background lawn and no reduction in the number of revertants/plate. The S9 optimization study was performed using TA98 and TA100 with the highest non-cytotoxic dose of test article, (10,000 ug/plate) and concentrations of S9 mix of 25-400 µl. In the absence of any effect 25 µl S9 mix/plate was used in the mutagenicity study. The test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However after incubation test material was visible at all dose levels in the top layer. The test material was not cytotoxic to any tester strain. In the repeat study statistically significant increases in revertant colonies were observed in TA1535 without metabolic activation and in WP2uvrA with metabolic activation. However since these findings were not found during the first experiment they were not considered biologically significant. The positive control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response. Dosing solution analysis confirmed that high dose concentration was acceptable.

Conclusions:
Interpretation of results (migrated information):
negative

The test material was not mutagenic to Salmonella and E. Coli strains with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium and E. coli WP2uvrA were exposed to an analogue of C20-C24 alkaryl calcium salt derivative (Analogue of CAS 70024 -69 -0), at concentrations of 100, 330, 1000, 3330 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (US-EPA, 1989, Ames Test according to OECD 471). A Dose Range-finding Study was conducted using tester strains TA98 and TA100, and dose levels of test material ranging from 0.003 to 10 mg/plate were used. No cytotoxicity was observed in the dose range-finding study with tester strains TA100 and WP2uvrA with or without metabolic activation as evidenced by normal background lawn and no reduction in the number of revertants/plate. The S9 optimization study was performed using TA98 and TA100 with the highest non-cytotoxic dose of test article, (10,000 µg/plate) and concentrations of S9 mix of 25400 µl. In the absence of any effect 25 µl S9 mix/plate was used in the mutagenicity study. In the main study there were two treatment sets for each tester strain, with (+S9) and without (-S9) metabolic activation. Each of the tester strains was dosed with five concentrations of test substance, vehicle controls, and a positive control. Three plates/dose group/strain/treatment set were evaluated. The results of the initial assay were confirmed in a second independent experiment. 100 µl of test material, positive control or vehicle control were added to each plate along with 100 µl of tester strain, S9 mix (if needed) and 2.0 ml of top agar. This was overlaid onto the surface of 25 ml minimal bottom agar in a petri dish. Plates were incubated for 48 hours at 37oC. The condition of the bacterial background lawn was evaluated for cytotoxicity and test article precipitate. The test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However after incubation test material was visible at all dose levels in the top layer. The test material was not cytotoxic to any tester strain. In the repeat study statistically significant increases in revertant colonies were observed in TA1535 without metabolic activation and in WP2uvrA with metabolic activation. However since these findings were not found during the first experiment they were not considered biologically significant. The positive control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response. Dosing solution analysis confirmed that high dose concentration was acceptable. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP, and therefore meets the criteria for Klimisch code 1. However, as this study report is used in a read-across approach, Klimisch 2 is assigned.
Qualifier:
according to
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)
Details on mammalian cell type (if applicable):
Clone tested: WBL
Cells were thawed and cultured in McCoy's 5A Medium containing 10% fetal bovine serum and 2 mM L-glutamine at 37oC, in 4-6% CO2 in air. Cultures were seeded at 1.2 x 10E6 cells (16-hour harvest) and 0.8 x 10E6 (40-hour harvest) approximately 1 day prior to dosing. Fetal bovine serum was excluded from activated culture
Metabolic activation:
with and without
Metabolic activation system:
S9 aroclor treated rats
Test concentrations with justification for top dose:
10, 20, 40, 80, 120, 160 µg/ml (50 µL sample was evaluated with and without metabolic activation).
Vehicle / solvent:
Tetrahydrofuran was used for the test article and acetone for positive control.
Vehicle and Positive Control concentration levels by activation status:
Acetone, 5 µg/mL with and without activation
Tetrahydrofuran, 5 µg/mL with and without activation
N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 µg/mL without activation
7,12-Dimethylbenz[a]anthracene (DMBA), 10 µg/mL with activation
Positive controls:
yes
Positive control substance:
other: Acetone (5 µg/ml (with and without activation
Positive controls:
yes
Positive control substance:
other: Tetrahydrofuran, 5 µg/mL (with and without activation)
Positive controls:
yes
Positive control substance:
other: N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 0.6 µg/mL (without activation)
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
10 µg/mL (with activation)
Details on test system and experimental conditions:
Exposure Method: Dilution
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 16 and 40 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 metaphase cells (100 per culture), each containing 19-223 chromosomes.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
Chromosome aberrations, either chromosome or chromatid type were recorded. Total aberration frequency, gaps, polyploid and endoreduplicated cells, pulverised chromosomes, Robertsonian translocations, translocations and abnormal monocentric chromosomes were recorded and excluded.
Statistics:
The number of cells with at least one aberrant chromosome and the number of cells examined in each replicate were used for statistical analysis. The number of aberrant individual chromosomes/cell was not analysed. Positive control groups were compared to vehicle control by Fisher Exact Test. Each pair of replicates was compared by Fisher Exact Test. Differences between control and treated groups were compared using Fisher Exact Test and if necessary a 2x2 Fisher Tests. A permutation test was performed to test for dose related trends. Significance levels of less than 0.05 were reported.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate and/or cloudiness were present with and without metabolic activation at concentrations of 80 µg/ml and greater.


RANGE-FINDING/SCREENING STUDIES: A grater than 50% reduction in cell counts or mitotic activity was not observed at concentrations up to 160 µg/ml.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cell survival was not significantly reduced when compared with the vehicle control in the screening study. Cell survival was reduced by at least 50% compared with the vehicle control in the repeat assay (40 hour harvest) without metabolic activation at the 160 µg/ml concentration. A greater than 50% reduction in mitotic index was not observed in either the initial or repeat assays at any other concentration tested.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

There were no statistically significant differences in the number of chromosomal aberrations at 16 hours with activation and at 40 hours with and without metabolic activation. In the initial 16 hour harvest with and without activation, a statistically significant increase was observed with one dose level different from the vehicle control. However, this finding was not evident in the repeat 16 hour harvest without activation. The observed initial increase was not reproducible and was not considered biologically significant. Positive and vehicle control group responses were as expected.

Remarks

In the culture medium solubility test precipitate and/or cloudiness were present with and without metabolic activation at concentrations of 39 µg/mL and 78 µg/mL and greater. In the pretest toxicity assay, a greater than 50% reduction in cell counts or mitotic activity was not observed at concentrations up to 160 µg/mL. The doses selected for the initial assay were 10, 20, 40, 80, 120 and 160 µg/mL. Cell survival was not significantly reduced when compared to the vehicle control in the initial assay. Cell survival was reduced by at least 50% compared to vehicle control in the repeat assay (40-hr harvest) without metabolic activation at the 160 µg/mL concentration. A greater than 50% reduction in mitotic index was not observed in either the initial or repeat assays at any concentration tested. Precipitation was observed at concentrations greater than 80 µg/mL in the chromosomal aberration assay. Therefore, the highest concentration evaluated at 16 hours was 80 µg/mL. There were no statistically significant differences in the number of chromosomal aberrations at 16 hours with activation and at 40 hours with and without metabolic activation. In the initial 16-hour harvest without activation a statistically significant increase was observed with one dose level different from the vehicle control. However this finding was not evident in the repeat 16-hour harvest without activation. The observed initial increase was not reproducible and was not considered biologically significant. Positive and vehicle control group responses were as expected. The positive control group had a statistically significant higher percentage of aberrant cells than the vehicle control group with and without activation at each harvest interval.

Conclusions:
Interpretation of results (migrated information):
negative

Not clastogenic under the terms of the study.
Executive summary:

In a mammalian cell cytogenetics assay [chromosome aberration], CHO cell cultures were exposed to an alkaryl magnesium salt derivative at concentrations of 0, 10, 20, 40, 80, 120 and 160 µg/ml with and without S9 metabolic activation. Positive controls induced the appropriate response.  There was no evidence of chromosome aberrations induced over background. This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data. 

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: study meets the criteria for Klimisch code 1. However as the study is used in a read-across approach, Klimisch 2 is assigned.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 aroclor induced rat liver
Test concentrations with justification for top dose:
62.5, 125, 250, 500, 1000 µg/plate
Vehicle / solvent:
tetrahydrofuran for test material
DMSO for positive control
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran 25 uL/plate, Dimethylsulfoxide 100 uL/plate
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
other: 2-Aminoanthracine (2AA) and N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG)
Remarks:
9-Aminoacridine (9AA), 100 µg/plate - TA1537 without S9; 2-Aminoanthracine (2AA), 2.5 µg/plate-all strains with S9; N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), 10 µg/plate-TA100, TA1535 without S9; 2-Nitrofluorene (2NF), 5 µg/plate-TA98, TA1538 without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Evaluation criteria:
number of revertant colonies
Statistics:
mean revertant colony number and standard deviation were determined for each dose point.
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed on plates with 5000 and 2000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
In the range finding study toxicity (notable reduction in background lawn and/or 50% reduction in the number of revertant colonies compared to vehicle control) was not observed at any concentration tested with or without metabolic activation. However the 5000 and 2000 µg/plate levels were difficult to evaluate due to test substance interference. At 1000 and 500 µg/plate precipitate was observed on the plates. These findings resulted in the selection of concentrations ranging from 62.5 to 1000 µg/plate for the main study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test substance did not induce significant increases in revertant colonies (equal to or greater than three times the THF control) in any of the tester strains, at any dose level, with or without metabolic activation in the initial or repeat assays. A greater than 50% reduction in mean number of revertant colonies compared to THF were observed in the initial assay in TA1537 without activation at 250 µg/plate. In TA1535 with activation, no background /no revertants was noted in the initial assay for all three plates at 250 and 500 and in two plates at 1000 µg/plate. The significance of these reductions is difficult to interpret since the findings were inconsistent between assays and dose levels. Precipitate was seen on all plates at 1000 µg/plate (+/- S9) in the initial and repeat assays. The positive controls produced at least a three-fold increase in revertant colonies compared with the DMSO control in their respective strains. Nontreated and vehicle controls were acceptable and were consistent with data from previous assays. The 1000 µg/plate concentration of test substance in THF was evaluated analytically for stability, concentration and homogeneity. Analysis conformed that the test substance was stable and homogeneous in THF for the intended period of use. The 1000 µg/plate solution was prepared and assayed twice during the study. The result for the first preparation was 121% above nominal. The result of the second preparation was 15% above nominal.

Conclusions:
Interpretation of results (migrated information):
negative

The test substance was not mutagenic in any strain of S typhimurium tested, including at least one dose above the solubility of the test substance. The genotoxicity NOEL was 1000 µg/plate.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium were exposed to a petroleum based calcium salt derivative, at concentrations of 6.25, 125, 250, 500 and 1000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence of induced mutant colonies over background. This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

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

Genetic toxicity in vivo

Description of key information

Micronucleus test, OECD 474, CAS 70024-69-0, negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP, and therefore meets the criteria for Klimisch code 1. However, as the study is used in a read-across approach, Klimisch 2 is assigned.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Male and female swiss albino Crl: CD-1 (ICR) BR aged 50 days at initiation.
Route of administration:
intraperitoneal
Vehicle:
peanut oil
Details on exposure:
single dose via intraperitoneal injection followed by a 72 hr evaluation period
Dose volume: 5 ml/kg
Duration of treatment / exposure:
72 hours
Frequency of treatment:
Once
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
200 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
400 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Peanut oil vehicle control: 18/sex;
triethylenemelamine positive control: 0.25 mg/kg, 5/sex;
100 and 500 mg/kg: 15/sex;
200 and 400 mg/kg: 18/sex
Control animals:
yes
Positive control(s):
triethylenemelamine positive control: 0.25 mg/kg, 5/sex; 100 and 500 mg/kg: l5/sex; 200 and 400 mg/kg: l8/sex
Tissues and cell types examined:
erythrocytes evaluated for micronuclei and ratio of non chromatic and polychromatic determined
Evaluation criteria:
NCE/PCE ratio and % PCE versus total erythrocytes
Statistics:
Animal to animal variability in spontaneous frequency of micronucleated polychromatic erythrocytes were evaluated in vehicle controls. Statistically
significant differences were evaluated in the frequency of micronucleated polychromatic erythrocytes between treated groups and vehicle controls.
NCE/PCE (normochromatic erythrocytes/polychromatic erythrocytes) ratios in treated and control groups were compared. Tests included dispersion test of AmpWett and Delow, and Margolin, Fishers exact test, binomial approximation, Cochran-Artage test for trend, a one-way analysis of variance and Dunett's procedure.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

During the main study, toxicity was observed at 400 and 500 mg/kg. at 500 mg/kg, 5 males and 4 females of 15/sex died prior to the scheduled sampling time. At 400 mg/kg, 1 of 18 treated females died on Day 3. Other clinical signs of toxicity included palpebral closure, decreased motor activity and weakness. Cytotoxicity was observed in both sexes. A statistically significant increase in NCE/PCE ratio was observed in individual animals of both sexes in other groups. Altered proportions of erythrocytes to nucleated cells were noted for both sexes in the treated groups. No biological or statistical significant increase in the number of micronucleated PCE/100 PCE expected for control animals. The variability in response observed in the vehicle controls. The positive control exhibited a statistically significant increase in micronuclei as expected. Chemical analysis confirmed that the dosing solution preparation procedure utilized for this study resulted in homogeneous solutions of appropriate concentration.

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study the test substance was not genotoxic. Under the conditions of this study the test material did not induce micronuclei in bone marrow erythrocytes of mice. The genotoxicity NOEL was 500 mg/kg.
Executive summary:

In a mouse bone marrow micronucleus assay, animals were treated via the peritoneum with C20-24 alkaryl calcium salt derivatives at doses of 0, 100, 200, 400 and 500 mg/kg bw.  Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.  The vehicle was peanut oil. There were signs of toxicity during the study at 400 and 500 mg/kg.  The positive control induced the appropriate response.  There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: study meets criteria for Klimisch code 1. However, as this study is used in a read-across approach, Klimisch 2 is assigned.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
Swiss albino CD 1 mice, 10-12 weeks at initiation
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
three treatments 24h apart followed by a 24h holding period prior to bone marrow sample collection.
Dose volume: 10 ml/kg
Duration of treatment / exposure:
three treatments 24h apart followed by a 24h holding period prior to bone marrow sample collection.
Frequency of treatment:
three
Post exposure period:
24h
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
cyclophosphamide
Tissues and cell types examined:
bone marrow, polychromatic erythrocytes
Evaluation criteria:
number of micronuclei / 1000 PCE
Statistics:
ANOVA, Duncan's multiple range, Wilk's criterion or Kolomogorov-Smirnov.
nonparametric analysis by Kruskal-Wallis and Dunns test, Joncheeres test of ordered response.

The following parameters were recorded and evaluated; the ratio of polychromatic to normochromatic erythrocytes, number of polychromatic erythrocytes with micronuclei and number of polychromatic erythrocytes scored. Statistical analysis included means and standard deviations of the micronuclei data and a test of equality of group means. Tests included a one-way analysis of variance, Duncan’s Multiple Range test and regression analysis. Residuals from the ANOVA were analyzed by Wilk’s Criterion or the Kolomogorov-Smirnov statistic. Nonparametric analyses included the Kruskal-Wallis one way ANOVA followed by Dunn’s Summed Rank Test. Dose response was evaluated by Jonkheere’s test of ordered response.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
All dose range finding animals survived and were free of clinical signs. Bone marrow toxicity was not observed at any dose levels tested. Therefore 2000 mg/kg was selected as the high dose for the micronucleus assay. The mid and low doses were selected to be 1/2 and 1/4 of the high dose.

RESULTS OF DEFINITIVE STUDY
All animals survived to scheduled sacrifice and were free of clinical signs. The responses of the vehicle control and positive control groups were appropriate and support the validity of the assay results. The positive control induced a significant increase in mean number of micronucleated polychromatic erythrocytes. In addition it induced cytotoxicity. There were no dose-related increases or statistical differences in micronuclei formation observed at any dose level of the test material. Cytotoxicity was not observed. There were no statistical decreases in the percentage of polychromatic erythrocytes compared to the vehicle control. Chemical analysis of dosing solutions confirmed that they were homogeneously prepared at the desired concentrations and that they were stable for the intended period of use.
Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the study the test substance was not genotoxic. Under the conditions of this study the test material did not induce micronuclei in bone marrow erythrocytes and did not induce cytotoxicity in the bone marrow of CD-1 mice. The genotoxicity NOEL was 2000 mg/kg.
Executive summary:

In a mouse bone marrow micronucleus assay, animals were treated via the peritoneum with C20-24 alkaryl calcium salt derivatives at doses of 0, 500, 1000, 2000 mg/kg bw.  Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.  The vehicle was peanut oil. The positive control induced the appropriate response.  There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

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

Additional information

Genetic toxicity data (in vitro) for read across substances

In the key study, a reverse gene mutation assay in bacteria, the strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium and E. coli WP2uvrA were exposed to a calcium sulfonate read across substance, (Analogue of CAS 70024 -69 -0), at concentrations of 100, 330, 1000, 3330 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (Sanitised, F., 1989, OECD 471). A Dose Range-finding Study was conducted using tester strains TA98 and TA100, and dose levels of test material ranging from 0.003 to 10 mg/plate were used. No cytotoxicity was observed in the dose range-finding study with tester strains TA100 and WP2uvrA with or without metabolic activation as evidenced by normal background lawn and no reduction in the number of revertants/plate. The S9 optimization study was performed using TA98 and TA100 with the highest non-cytotoxic dose of test article, (10,000 µg/plate) and concentrations of S9 mix of 25-400 µl. In the absence of any effect 25 µl S9 mix/plate was used in the mutagenicity study. In the main study there were two treatment sets for each tester strain, with (+S9) and without (-S9) metabolic activation. Each of the tester strains was dosed with five concentrations of test substance, vehicle controls, and a positive control. Three plates/dose group/strain/treatment set were evaluated. The results of the initial assay were confirmed in a second independent experiment. 100 µl of test material, positive control or vehicle control were added to each plate along with 100 µl of tester strain, S9 mix (if needed) and 2.0 ml of top agar. This was overlaid onto the surface of 25 ml minimal bottom agar in a petri dish. Plates were incubated for 48 hours at 37°C. The condition of the bacterial background lawn was evaluated for cytotoxicity and test article precipitate. The test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However after incubation test material was visible at all dose levels in the top layer. The test material was not cytotoxic to any tester strain. In the repeat study statistically significant increases in revertant colonies were observed in TA1535 without metabolic activation and in WP2uvrA with metabolic activation. However since these findings were not found during the first experiment they were not considered biologically significant. The positive control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response. Dosing solution analysis confirmed that high dose concentration was acceptable. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

In a supporting study with a magnesium sulfonate read across substance, (CAS 71786-47-5), the test material was applied (doses of 62.5, 125, 250, 500 and 1000 µ/plate) in agar to the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (Sanitised, I., 1995). As metabolic activation system, S9 aroclor induced rat liver was used. Tetrahydrofuran was the vehicle for the test material, and DMSO was the vehicle for the positive controls (9-aminoacridine, 2-nitrofluorene and 2-aminoanthracene and N-Methyl--N-Nitro-N-Nitrosoguanidine). The test substance did not induce significant increases in revertant colonies (equal to or greater than three times the THF control) in any of the tester strains, at any dose level, with or without metabolic activation in the initial or repeat assays.

Chromosome abberation data (in vitro) for read across substances

In the key study, a mammalian cell cytogenetics assay [chromosome aberration], Chinese hamster ovary (CHO, clone tested WBL) cell cultures were exposed to a magnesium sulfonate red across substance, (CAS 71786-47-5), at concentrations of 0, 10, 20, 40, 80, 120 and 160 µg/ml with and without S9 metabolic activation (Sanitised, J., 1995, according to OECD 473). As vehicle Tetrahydrofuran (THF, for the test material) and acetone (for the positive controls) was used. The solubility of the test substance in the culture medium was established at concentrations of 20, 39, 78, 156, 3 13, 625, 1250, and 2500 µg/ml. Visual and microscopic examinations were made for precipitation at 0, 30 and 180 minutes post preparation. Concentrations showing signs of insolubility at any of these time points were considered unsuitable for dosing. A Dose range finding study was conducted with the test substance and vehicle controls tested in duplicate cultures each with and without activation. Test substance tested at concentrations of 2.5, 5, 10, 20, 40, 60, 80, 120, and 160 µg/ml. Cytotoxicity and mitotic indices were evaluated. In the main study there were two treatment sets for each concentration of test substance, with (+S9) and without (-S9) metabolic activation. 7,12-Dimethylbenz(a)anthracene - DMBA (positive control) was tested with activation and N-Methyl-N-Nitro-N-Nitrosoguanidine - MNNG (positive control) was tested without activation. Prepared cultures were treated with test substance or control material and were incubated for 16 hours. A repeat assay was performed using 16 and 40 hour harvest time points. Vehicle, MNNG and DMBA cultures were incubated for 16 hours only. Two to three hours prior to the 16 and 40 hour harvest the spindle inhibitor, Colcemid, was added to each culture to obtain a final concentration of 0.2 µg/ml. Harvested cells were evaluated microscopically for per cent confluency, morphology and estimated number of mitotic cells prior to harvest. The initial assay and the repeat assay (each after 16-hr incubations) with and without S9 were conducted with 20, 40 and 80 µh/ml. The repeat assay with and without S9 (after 40-hr incubations) was conducted with 80, 120 and 160 µg/ml. Slides were prepared for these groups using Giemsa stain. Two slides/treatment groups were evaluated. 200 metaphase cells (100 per culture) each containing 19-23 chromosomes per treatment group were scored. Chromosomes were counted for each cell. Chromosome aberrations, either chromosome or chromatid type were recorded. The following observations were recorded and excluded from the total aberration frequency: gaps, polyploid and endoreduplicated cells, pulverized chromosomes, Robertsonian translocations, translocations and abnormal monocentric chromosomes. The percentage of aberrant cells and the frequency of aberration (%) per treatment group were determined. In order for a test substance to be considered to have induced a positive response compared to vehicle control a statistically significant dose related increase in the percentage of aberrant cells along with a mean percentage of aberrant cells in excess of 5% in at least one treatment group were required. Or, a reproducible and statistically significant response in at least one treatment group with a mean % of aberrant cells exceeding 5% was observed. Test substance concentration verification was performed on the highest stock concentration in both the initial and repeated assays.

Results were within 8% of nominal. In the culture medium solubility test precipitate and/or cloudiness were present with and without metabolic activation at concentrations of 39 µg/ml and 78 µg/ml and greater. In the pretest toxicity assay, a greater than 50% reduction in cell counts or mitotic activity was not observed at concentrations up to 160 µg/ml. The doses selected for the initial assay were 10, 20, 40, 80, 120 and 160 µg/ml. Cell survival was not significantly reduced when compared to the vehicle control in the initial assay. Cell survival was reduced by at least 50% compared to vehicle control in the repeat assay (40-hr harvest) without metabolic activation at the 160 µg/ml concentration. A greater than 50% reduction in mitotic index was not observed in either the initial or repeat assays at any concentration tested. Precipitation was observed at concentrations greater than 80 µg/ml in the chromosomal aberration assay. Therefore, the highest concentration evaluated at 16 hours was 80 µg/ml. There were no statistically significant differences in the number of chromosomal aberrations at 16 hours with activation and at 40 hours with and without metabolic activation. In the initial 16-hr harvest without activation a statistically significant increase was observed with one dose level different from the vehicle control. However this finding was not evident in the repeat 16-hr harvest without activation. The observed initial increase was not reproducible and was not considered biologically significant. Positive and vehicle control group responses were as expected. The positive control group had a statistically significant higher percentage of aberrant cells than the vehicle control group with and without activation at each harvest interval. Positive controls (N-Methyl-N-Nitro-N-Nitrosoguanidine without activation and 7,12-Dimethylbenz(a)anthracene DMBA with activation) induced the appropriate response. There was no evidence of chromosome aberrations induced over background. Therefore, the test substance is not considered not mutagenic or genotoxic in this assay with and without metabolic activation under the conditions of this study.

Data on DNA damage and/or repair - micronucleus assay for read across substances

In the key study, a mouse bone marrow micronucleus assay, Swiss Albino Crl:CD-I (ICR) BR mice were treated once via the peritoneum with a calcium sulfonate read across substance, Analogue of CAS 70024 -69 -0, at doses of 0, 100, 200, 400 and 500 mg/kg bw (Sanitised, G., 1989, OECD 474). Bone marrow cells were harvested at 24, 48 and 72 hours post-treatment.  A range-finding study was conducted at 200,400 and 600 mg/kg. Mortality and physical observations were evaluated. During the dose range-finding study mortality (9 of 10 animals) was observed at 600 mg/kg but not at lower dose levels. Signs of toxicity observed at all dose levels included reduced faeces, reduced food consumption, hyperactivity and phonation. Decreased motor activity was observed at 400 and 600 mg/kg. Based on these results dose levels of 100, 200, 400 and 500 mg/kg were selected for the main study. In the main study the vehicle was peanut oil and the dose volume 5 mL/kg. As positive control substance triethylenemelamine was used. All animals were observed frequently for physiological or behavioural abnormalities on the day of dosing and at least twice daily thereafter. Body weights were taken on the first day of the study prior to treatment and at sacrifice. Macroscopic pathology performed on all animals at sacrifice. Five/sex from each treatment group and vehicle control group were sacrificed for bone marrow sampling 24, 48 and 72 hours post treatment. Positive controls sampled at 24 hours only. NCE/PCE ratio and %PCE of total erythrocytes were calculated by counting a total of > 1000 erythrocytes/animal. A total of 1000 PCE /animal were evaluated for the presence of micronuclei. The number of micronuclei in NCEs was also determined.

During the main study, toxicity was observed at 400 and 500 mg/kg. At 500 mg/kg 5 males and 4 females of 15/sex died prior to the scheduled sampling time. At 400 mg/kg 1 of 18 treated females died on day 3. Other clinical signs of toxicity included palpebral closure, decreased motor activity and weakness. Cytotoxicity was observed in both sexes. A statistically significant increase in NCE/PCE ratio was observed in males at 500 mg/kg at 24 hours. Elevated ratios were also observed in individual animals of both sexes in other groups. Altered proportions of erythrocytes to nucleated cells were noted for both sexes in the treated groups. No biological or statistical significant increase in the number of micronucleated-PCE was observed in any treated group compared to the vehicle control. All values for individual animals were within the expected range of micronucleated-PCE/1000 PCE expected for control animals. The variability in response observed in the treated animals was similar to that observed in the vehicle controls. The positive control exhibited a statistically significant increase in micronuclei as expected. Chemical analysis confirmed that the dosing solution preparation procedure utilized for this study resulted in homogeneous solutions of appropriate concentration. As there was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time, under the conditions of this study the test material did not induce micronuclei in bone marrow erythrocytes of mice.

This result is supported by a supporting study with a magenesium sulfonate read across substance, (CAS 71786-47-5), that showed no induction of micronuclei (Sanitised, K., 1995, CAS 71786-47-5, OECD 474, negative). The test material was administered in peanutoil via oral gavage (0, 500, 1000 and 2000 mg/kg bw) to swiss albino CD-1 mice (5 animals). Three treatments, each 24h apart followed by a 24h holding period prior to bone marrow sample collection were conducted with a dosing volume of 10 ml/kg. As positive control cyclophosphamide was used. All animals were observed for viability twice daily during the dosing period. Detailed clinical observations were recorded after each test substance administration. Body weights were recorded prior to initiation of dosing. Twenty-four hours after the third dose the animals were sacrificed for bone marrow sampling. Necropsies were not performed. A total of 2000 polychromatic erythrocytes/animal were evaluated for the presence of micronuclei. The per cent of PCE in the total population of erythrocytes was determined for each animal by counting the total polychromatic and normochromatic erythrocytes.

All animals survived to scheduled sacrifice and were free of clinical signs. The responses of the vehicle control and positive control groups were appropriate and support the validity of the assay results. The positive control induced a significant increase in mean number of micronucleated polychromatic erythrocytes. In addition it induced cytotoxicity. There were no dose-related increases or statistical differences in micronuclei formation observed at any dose level of the test material. Cytotoxicity was not observed. There were no statistical decreases in the percentage of polychromatic erythrocytes compared to the vehicle controls.


Justification for selection of genetic toxicity endpoint
well documented GLP-guideline study


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the all in vitro and in vivo data available for the substance, it is clear that the magnesium and calcium sulfonate read across substances are neither mutagenic, nor genotoxic, nor induced micronuclei. Therefore also the magnesium sulfonate target substance is considered to be not mutagenic nor genotoxic. According to the European regulation (EC) No. 1272/2008 the test material does not meet the criteria for classification and will not require labelling as a mutagen.