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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
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.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and 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 in ethanol
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
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 for each dose point.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
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
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No reduction in the number of revertants/plate was observed in the rangefinding study with strains TA100 and WP2uvrA.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in the dose rangefinding study with strains TA100 and WP2uvrA with or without metabolic activation.

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

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

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 analog of C20-C24 alkaryl calcium salt derivative, at concentrations of 100, 330, 1000, 3330 and 10000 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:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP, so therefore meets the criteria of Klimisch code 1.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK +/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 cells
Test concentrations with justification for top dose:
500, 1000, 1500, 2000, 4000, and 5000 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with activation
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without activation
Details on test system and experimental conditions:
The test material was suspended in culture and palced on a restricted media containing trifluorothymidine.
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 4 hours
- Exposure duration: 10-12 days

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Number of colonies/plate.
Statistics:
Mean and standard deviation.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Significant toxicity (<90% total growth) at 500 µg/ml occurred both with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test material was not genotoxic under the conditions of test.
Executive summary:

In a mammalian cell gene mutation assay, mouse lymphoma L5178Y cells cultured in vitro were exposed to a petroleum derived calcium salt, at concentrations of  500, 1000, 1500, 2000, 4000 and 5000 µg/ml in the presence and absence of mammalian metabolic activation. 

 

The positive controls induced the appropriate response.  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 476 for in vitro mutagenicity (mammalian forward gene mutation) data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP, and therefore meets the criteria for Klimisch code 1.
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):
Not specified
Additional strain / cell type characteristics:
not specified
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
Vehicle / solvent:
Tetrahydrofuran was used for the test article and acetone for positive control.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
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:
Fisher exact test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate and/or cloudiness were present with and without metabolic activationat 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 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.

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.

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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

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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:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and to GLP, and therefore meets the criteria for Klimisch code 1.
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
Duration of treatment / exposure:
72 hours
Frequency of treatment:
Once
Remarks:
Doses / Concentrations:
0, 100,200,400 and 500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
trethylenemelame 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 eryocytes were evaluated in vehicle controls. Statistically signficant differences were evaluated in the frequency of micronucleated polychromatic eryhrocytes between treated groups and vehicle controls.
NCE/PCE (normochromatic eryhrocytes/polychromatic eryhrocytes) 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 dies 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 ths study the test substance was not genotoxic.
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 cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start Date of Experimental Work: October 28,1987 Completion Date of Experimental Work: December 14, 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with some minor deviations from standard test guidelines and/or minor methodological deficiencies, which limit the reliability of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
other: The study was conducted in compliance with the Good Laboratory Practices (GLP) regulations of the U.S. Environmental Protection Agency (EPA), (40 CFR, Part 792) and according to the protocol and. standard operating procedures.
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
Strain B6C3Fl mice were used in the study. Male and female mice were obtained from a reputable supplier. The males were dosed when they were 13 weeks old while the females were dosed when they were 12-14 weeks old. Mice were quarantined at least one week and were group housed in cages with Easi-litter bedding. All animals were fed Agway Prolab R-MH 3000. They were given untreated Cambridge tap water supplied ad libitum. Animals were housed in a room with automatic light/dark cycle of approximately 12 hours each. Temperature of the environment was maintained at 72°F (± 10%). Each mouse was randomly assigned to code groups using computer generated random numbers, identified by ear punch, and individually caged following a quarantine period. Approximately eight hours prior to dosing feed was removed from the animals. One male mouse, 63, dosed with cyclophosphamide had some food in its cage at the time of dosing; but this did not affect the study since a positive result was observed in-this animal
Route of administration:
oral: gavage
Vehicle:
Due to difficulty in dissolving it in common vehicles such as saline or corn oil, the test material was administered to mice by oral gavage without use of a solvent
Details on exposure:
The induction of micronuclei in the bone marrow polychromatic erythrocyte cells as a result of chemical treatment is an established test for determining the genotoxic effect of chemicals. Micronuclei are cytoplasmic bodies with the appearance of small nuclei in the cell. These entities arise from chromosomal lagging at anaphase, from acentric chromosomal fragments, or from spindle fiber malformation during cell division. In mouse bone marrow, the cell population tested consists of erythroblasts undergoing their final mitosis before expulsion of the nucleus. The frequency of occurrence
of micronuclei in the polychromatic erythrocyte cells of treated animals provides an indicator of in vivo cytogenetic damage. Polychromatic erythrocytes or young red blood cells mature into normochromatic erythrocytes (NCEs) in the bone marrow. In normal untreated bone marrow cells, polychromatic erythrocytes and normochromatic erythrocytes are approximately in equal ratio. If a test agent is cytotoxic to nucleated ·cells, the proportion of polychromatic erythrocytes is reduced. The ratio of polychromatic erythrocytes to normochromatic erythrocyte is often used as an index of cytotoxicity of a test material.

A total of 80 mice, 40 males and 40 females, were used for the study.
Ten animals were used per test group. The animals were dosed once by oral gavage. Dosing volumes for each animal were determined from the weight of the animal, the specific gravity of the chemical, and the final dose desired. Dose volumes less than 0.1 ml were rounded to 0.1 ml. The target dose of the test chemical was 5 g/kg and the negative control was mineral oil at 5 ml/kg.

Time of Sacrifice (hours)
18 24 48
OS 68022C 10 10 10
Mineral Oil 10 10 10
Cyclophospahmide(50mg/kg) 10


Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
once only
Remarks:
Doses / Concentrations:

Basis:
other: neat 5g/kg
No. of animals per sex per dose:
A total of 80 mice, 40 males and 40 females, were used for the study.
Ten animals were used per test group 5 x male 5x female
Control animals:
yes
Positive control(s):
Cyclophosphamide 50mg/kg
Tissues and cell types examined:
Bone Marrow
Details of tissue and slide preparation:
Bone Marow Collection
Following each exposure period (18, 24 and 48 hours), mice were killed by cervical dislocation and the femurs were removed and the ends cut. Bone marrow cells were collected by flushing the cavity with a phosphate buffered saline solution (pH 7.4).

Slide preparation
After collection of the bone marrow, the suspension was centrifuged and the supernatant was removed. Cells were resuspended in phosphate buffered saline solution. Slides were made by placing one drop of suspension on a clean slide and spreading the cells evenly with a second slide. At least two slides were made from each animal. Slides were labelled with the case number, chemical number, animal group number, animal identification number and an alphabet letter to designate replicate slides. Slides were than air dried at least 24 hours and fixed in 100% methanol for five minutes. Slides from mice treated with the test substance, and the negative control were coded, and the code was recorded in the original data notebook.

Staining
Acridine orange stain supplied by Sigma Chemical Co. (Lot No. l24F-3708) was made at a final concentration of 0.125mg/ml in sterile phosphate buffer saline solution (pH 7.4). Slides were stained a few minutes prior to analysis using a fluorescent microscope.
Evaluation criteria:
For each animal a minimum of 1000 polychromatic erythrocytes (PCEs) were counted for the presence of micronucleated PCEs. The frequency of micronucleated cells per animal was expressed as the number of micronucleated PCEs per 1000 PCEs' counted. The ratio of PCEs to normochromatic erythrocytes (NCEs) in 1000 total erythrocytes for each animal was recorded. The data were analyzed for statistical significance based on a binomial distribution, a level of significance of 0.05, and the tables of Kastenbaum and Bowman (Mutat Res ~ 527-549, 1970)
Statistics:
The data were analyzed for statistical significance based on a binomial distribution, a level of significance of 0.05, and the tables of Kastenbaum and Bowman (Mutat Res ~ 527-549, 1970)
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
One male mouse, 63, dosed with cyclophosphamide had some food in its cage at the time of dosing; but this did not affect the study since a positive result was observed in-this animal.
Conclusions:
Interpretation of results (migrated information): negative
Chemical OS 68022C was tested for its genotoxicity using the mouse in vivo micronucleus screening assay. There was no significant increase in micronucleated PCEs in animals exposed to the test substance. Thus, OS 68022C was negative in this in vivo assay.
Executive summary:

Chemical OS 68022C was tested for its genotoxic activity using a mouse in vivo micronucleus screening assay. Male and female B6G3Fl mice were dosed once at 5 g/kg. The mice were killed at 18, 24 and 48 hours after dosing, and the bone marrow cells were collected. Ten animals, five males and five females were included in each group. At least one thousand polychromatic erythrocytes (PGEs) from each mouse were examined for micronuclei.

The negative control was mineral oil dosed at 5 ml/kg for 18, 24 and 48 hours, while the positive control was cyclophosphamide dosed at 50 mg/kg for 24 hours.

The mean micronucleated PGEs for negative control male mice were 2.6, 3.0 and 2.4 per 1000 PGEs for the three time periods. The average number of micronucleated PCEs for the positive control male mice was 14.5.

The mean micronuc1eated PCEs for the test chemical were 1.7, 4.3 and 3.6 per 1000 PCEs for the three time points, respectively. These means are not significantly higher than the negative control values.

The mean number of micronucleated PCEs for negative control female mice were 1.9, 2.1 and 2.9 per 1000 PCEs for the three time periods, while the positive control female mice had an average of 20.5. The frequencies of micronucleated PCEs in female mice treated with the test chemical were 2.9, 3.2 and 2.1 per 1000 PCEs for the three time points, respectively. These means are not significantly higher than the negative control values.

Based on these data, chemical OS 68022C which was tested at the maximum dose of 5 g/kg, did not induce a significant increase in micronuclei in bone marrow cells from either male or female B6G3Fl mice. Thus, the chemical was negative in this in vivo genetic toxicity test.

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

Additional information

All of the data available for the registered substance to assess the mutagenic, clastogenic and genotoxic effects of the substance in vitro and in vivo. All the studies achieved negative results.

Data have been included that were conducted on sodium and magnesium salts. The sodium and magnesium ions are considered likely to be more active than calcium and the data is therefore considered suitable as supporting data for the endpoint.

Short description of key information:

A number of genotoxicity studies have been conducted in vitro and in vivo to determine the genotoxic effects of the registered substance

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the in vitro and in vivo data available for the substance, the substance is considered to be not genotoxic.