7,7-dimethyl-3-oxa-6-azaoctan-1-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported in the data over 12 years old provided by ECHA.

Reliability:

other: Reliability of study not reported in the data over 12 years old provided by ECHA.

Qualifier:

according to guideline

Guideline:

other: Annex V

GLP compliance:

yes

Type of assay:

other: in vitro mammalian gene mutation (B17)

Species / strain / cell type:

mammalian cell line, other: Chinese hamster ovary

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 625...5000 µg/ml
Concentration range in the main test (without metabolic activation): 625...5000 µg/ml

Vehicle / solvent:

Distilled water

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 18 hours

Fixation time:
6th Amendment fixation time : 18

Species / strain:

Chinese hamster Ovary (CHO)

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:

Observations:
Positive control substances gave appropriate responses.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Negative.

Reference 2

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

Study period:

19 May 88 to 06 June 88

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK +/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Culture preparation: L5178Y TK +/- Mouse Lymphoma Cells were treated with methotrexate, thymidine, glycine and hypoxanthine prior to freezing to eliminate possible spontaneous TK -/- mutants. As needed, cells were then removed from liquid nitrogen storage, cultured and maintained in logarithmic phase for five days before use in the assay system.

Culture medium - F10p
Cloning medium - F20p

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver (S9 mix)

Test concentrations with justification for top dose:

Toxicity assay: Doses ranged from 1 to 1000 μg/ml.
Mutagenesis assay: Based on the results of the toxicity assay, six doses were used in the mutagenesis assay; 500, 700, 900, 1100, 1300 and 1500 μg/ml, but only five doses were plated. Because all doses had greater than 10% relative suspension growth, the lowest dose was eliminated; and 700, 900, 1100, 1300 and 1500 μg/ml were treated with and without activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle:Considered under the conditions of the assay to be stable for the duration of the assay.

The test material and positive controls were diluted and dosed prior to administration.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hour and 20 minute exposure
- Expression time (cells in growth medium): 2-day expression period


SELECTION AGENT (mutation assays): TFT = Trifluorothymidine Deoxyriboside


NUMBER OF REPLICATIONS: 3 plates per dose group.


NUMBER OF CELLS EVALUATED:
Approximately 9XE5 cells were plated in 30ml F20p with 2.5 μg/ml TFT. Concurrently, approximately 180 cells were plated in 30 ml F20p without a selective agent, to determine viable colony counts (VC).


DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growths, relative cloning growths, relative total growths, mutant frequencies amd induced mutant frequencies were calculated.

Plates were labelled with study number, compound identification number, the dose administered, VC or TFT, +S9 or -S9. Colonies were enumerated after eleven days incubation at 36-38 deg C with 4.5-5% CO2. Plate counts were made with a Biotran III colony counter and recorded.

Evaluation criteria:

A test material is considered positive if it yields a 2-fold increase over the spontaneous mutant frequency of vehicle controls at one or more dose levels, if a dose related increase in response is observed and if the relative total growth is greater than 10%. Should the relative total growth, (survival) be less than 10% and a two-fold increase in mutant frequency observed, the test material will be considered negative.

Statistics:

The following equations were used in the data analysis of the forward mutation assay:
Relative Suspension Growth, % =
Total suspension growth of test culture / mean total suspension growth of vehicle controls x 100%

Relative cloning growth, % =
Mean number of VC Colonies on “X” treated plates / Average of mean of VC colonies on vehicle control plates x 100%

Relative total growth, % =
(Relative suspension growth, %) (Relative cloning growth, %) / 100%

Mutant Frequency (mutants / 10E4) =
Mean number of TFT colonies for “X” / Mean number of VC colonies for “X” x 2

Induced Mutant Frequency (IMF) = Mutant frequency of treated cultures - mean mutant frequency of vehicle control cultures

VC = Viable colonies
"X" = Dose being evaluated

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:

Dose selection for the repeat mutation assay* was based on a toxicity (dose determination) assay. The doses selected were 700, 900, 1100, 1300 and 1500 μg/ml. These were tested with and without metabolic activation.

*The initial mutation assay was repeated because plate count values were out of range.

Please see attached background material for Table 1 - Summary of mouse lymphoma mutagenicity data, and Table 2 - Mouse growth results with and without metabloic activation.

A mutant frequency greater than 2(X) the mean control mutant frequency was not observed at any dose. The positive (DMBA and EMS) and negative (Controls 1 and 2, + and - S9) controls responded in a manner consistant with data from previos analysis.

Remarks on result:

other: strain/cell type: L5178Y cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

A mutant frequency that was greater than two times the mean vehicle control frequency was not observed at any dose level, nor was there any evidence of a dose related response.
For this reason, it can be concluded that, under the conditions of this assay, the test substance does not induce gene mutations in L5178Y mouse lymphoma cells.

Executive summary:

The mutagenic activity of the test substance was tested in the L5178Y mouse lymphoma assay. The positive controls were dimethylbenzanthracene (DMBA) and ethylmethanesulphonate (EMS). The substance was tested at 700, 900, 1100, 1300, 1500 μg/ml

with and without metabolic activation. Following a three hour and 20 minute exposure period, test and control cultures were allowed a two day expression period. Cultures were cloned in selective medium, then incubated for eleven days. Surviving and mutant colonies were then counted and the mutant frequencies calculated.

There was no evidence of a dose related increase in mutagenic frequency, and the mutant frequencies were not significantly different from control at any dose. For this reason, it can be concluded that, under the conditions of this assay, the test substance does not induce gene mutations in L5178Y mouse lymphoma cells.

The positive (DMBA, EMS) and negative (DMSO) controls responded in a manner consistant with data from previous studies.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 84 to 23 March 84

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Vehicle control criteria not met, please see results

Principles of method if other than guideline:

A retention sample for the vehicle/test material mix was not taken.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Hisitidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver (S9 mix)

Test concentrations with justification for top dose:

Toxicity pre-test:To determine the highest dose of compound to be used in the assay, a dose range from 1 to 2000 μg per plate was tested.

Mutagenesis assay: The dose range for the mutagenesis assay was determined to be 1000, 500, 100, 10 μg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9-mix: (BaP)

Positive controls:

yes

Positive control substance:

other: 2-aminoflurene (2-AF)

Remarks:

With S9-mix

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

With S9-mix: (MNNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: approx 18 - 24 hours
- Exposure duration: 2 - 3 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 - 3 days.


NUMBER OF REPLICATIONS: 3 plates per dose.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: After incubation all plates were evaluated for toxic effects and read either manually or with an automatic colony counter.


OTHER:
Experimental evaulation: Toxicity of Test Material
To determine the highest dose of compound to be used in the assay, a dose range from 1 to 2000 μg per plate was tested with strain TA98, with and without metabolic activation. A single plate per dose point was used and the conditions were identical to that for the regular assay. Colony counts were made at 3 days and plates were evaluated for toxic response. The highest dose selected for use in the assay was that dose immediately below the one which was overtly toxic for the organisms.

Evaluation criteria:

Any test value which was more than three times the mean value of the concurrent vehicle control was considered to be a positive result.

Statistics:

The mean plate count and standard deviation for each dose point were determined.

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:

cytotoxicity

Remarks:

Slight toxicity seen at 1000 μg

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxicity seen at 1000 μg

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A completely lethal response was not seen up to 2000 μg/plate in the toxicity pre-test. Slight toxicity, however, was seen as a reduction of background lawn on the 1000 μg and 2000 μg plates with and without metabolic activation and on the 500 μg plate without metabolic activation. The dose range for the mutagenesis assay was determined to be 1000, 500, 100, 10 μg per plate.


ADDITIONAL INFORMATION ON CYTOTOXICITY: None.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

In the mutagenesis assay negative responses were noted in all five tester strains.

The positive and negative controls responded in the appropriate manner, with the exeception of TA1538 and BaP +S9. TA1538 response to BaP did not exceed the 3X over vehicle control criteria, but did show significant increase (3.7X) over background to warrant to strain as acceptable.

Conclusions:

Interpretation of results:
negative

The test material did not demonstrate mutagenic activity in this test system. The positive and negative controls responded in an appropriate manner. The test substance did not induce a noticable increase in the mutation rate of the tester organisms, either alone or with metabolic activation derived from rat liver. It can be concluded that, under the conditions of the assay, this material is not mutagenic for the Salmonella tester strains at doses up to and including ones which were toxic for these organisms.

Executive summary:

This assay is a test for the ability of a compound to cause reverse mutation at the histidine loci in bacteria. Five special tester strains of Salmonella typhimurium, which are sensitive to frameshift or base pair mutagens, are employed. Test results are considered to provide presumptive evidence of mutagenic potential in mammalian species. Test doses for the assay are based upon results of a pretest for toxicity. The highest dose selected is one that s clearly toxic for the organisms, but less than a totally lethal dose.

The dose range for the mutagenesis assay was determined to be 1000, 500, 100, 10 μg per plate.

Under the conditions of the assay, the test substance did not induce mutation in any of the five tester strains, and is judged to be negative.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 14, 1983 to December 30, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary, line K1, ATCC CEL G1

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver (S9 mix)

Test concentrations with justification for top dose:

1.82 μg/ml, 18.2 μg/ml, 182 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 24 hours
- Exposure duration: 7.5 hours and 24 hours.
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours prior to harvest


SELECTION AGENT (mutation assays): Not applicable.
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa stain


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: Fifty metaphase spreads from each flask were evaluated.


DETERMINATION OF CYTOTOXICITY
- Method: Metaphase spreads were evaluated for the presence of any and all structural abnormalities.

Evaluation criteria:

Levels of damage that were above the accepted spontaneous damage level for this system would be considered a positive result.

Statistics:

For each treatment and control group the total and mean values for each type of aberration were calculated. Mean values, with standard deviation, were calculated for numbers of abnormal cells and numbers of abnormalities observed.

Species / strain:

Chinese hamster Ovary (CHO)

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: A toxicity test was performed with and without S9 activation, to determine which doses to use. An LD50 was observed at 250 μg/ml, and an LD0 was observed at 50 μg/ml. The LD30 was then calculated. A high dose of 182 μg/ml was used with and without activation. The intermediate and low doses were one tenth and one hundredth serial dilutions of the top dose (18.2 and 1.82 μg/ml)




COMPARISON WITH HISTORICAL CONTROL DATA: Historically, the spontaneous damage levels for non-treated or vehicle controls are 0.0% - 8.0%. All vehicle controls for this study fall within accepted values (2.00%-5.34%).


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test substance: All three dose levels, at both time periods, with and without metabolic activation showed types of damage and damage levels comparable to the vehicle and non-treated controls (2.00%-6.00%). These values fall well within historical spontaneous damage levels, observed in this laboratory.

Cyclophosphamide: The positive controls showed damage levels of 21.00%.

Nontreated control plus activation: The nontreated controls showed damage levels of 5.00%.

Nontreated control w/o activation: The nontreated controls showed damage levels of 2.00%

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 examined for its ability to cause chromosomal mutations in CHinese Hamster Ovary cells when dosed at 182, 18.2 and 1.82 μg/ml with and without metabolic activation for 7.7 and 24 hours. The levels of damage observed were comparable to the controls and are well within the historical control data, or no effect, values.

With the framework of the test system, the test substance appears to be incapable of cause cytogenetic damage to CHO cells.

Executive summary:

The cytogenetic activity of the test substance was evaluated in Chinese Hamster Ovary (CHO) cells, grown in vitro. The test material was administered in the culture medium at 182, 18.2 and 1.82  μg/ml with and without S9 activation. Test and control flasks were divided into two groups, harvested at about 7.5 and 24 hours following a one hour dosing period. Approximately two hours prior to harvest all flasks received 1  μg/ml of colcemid, to induce metaphase arrest in the cells. The metaphase cells were harvested and prepared for cytogenetic evaluation. Fifty metaphase spreads from each flask were evaluated, under code to avoid bias, for the presence of any and all structural abnormalities.

The cytogenetic evaulation revealed no levels of damage that were above the accepted spontaneous damage level for the system (less than 8.00%). The positive controls, however, showed a marked increase in chromosomal abnormalities (21.00%).

Within the framework of this test system the test substance was not found to cause increased chromosal damage.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported in the data over 12 years old provided by ECHA.

Reliability:

other: Reliability of study not reported in the data over 12 years old provided by ECHA.

Qualifier:

according to guideline

Guideline:

other: Annex V

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: bacteria: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 0 - 5000 μg/plate
Concentration range in the main test (without metabolic activation): 0 - 5000 μg/plate

Vehicle / solvent:

Solvent: Water

Species / strain:

other: bacteria: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 μg/plate)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: bacteria: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 μg/plate)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: bacteria: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 μg/plate)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: bacteria: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 μg/plate)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Observations: No toxicity was observed.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The substance is not mutagenic.

Reference 6

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:

Not reported in the data over 12 years old provided by ECHA.

Reliability:

other: Reliability of study not reported in the data over 12 years old provided by ECHA.

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

No data.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 6 - 3200 μg/ml
Concentration range in the main test (without metabolic activation): 6 - 3200 μg/ml

Vehicle / solvent:

Distilled water.

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 21 hours

Fixation time:
6th Amendment fixation time: 21 hours

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>3200 μg/ml)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>3200 μg/ml)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>3200 μg/ml)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(>3200 μg/ml)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Observations: No effect on mitotic index at all concentrations tested.

Metaphase analysis was carried out only on concentrations at and above 400 micrograms/ml. No increase in aberrations were seen.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The substance was not found to cause increased chromosal damage.

Genetic toxicity in vivo

Description of key information

Bacterial Reverse Mutation Assay = Negative; OECD 471; Traul (1984) In Vitro Mammalian Chromosome Aberration Test = Negative; OECD 473; Traul (1983) In Vitro Mammalian Cell Gene Mutation Assay = Negative; OECD 476; Przydoda (1988) In Vivo Mammalian Erythrocyte Micronucleus Test = Negative; OECD 474; Przygoda (1988)

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc, Kingston, N.Y 12484
- Age at study initiation: Approx 9-10
- Weight at study initiation: 20 -40 grams at initiation
- Assigned to test groups randomly: yes - study animals were selected using a computer generated, body weight sorting program.
- Housing: Suspended stainless steel cage (after a week of group housing all animals were then single housed)
- Diet (e.g. ad libitum): Purina Certified Chow (pellets), ad libitum
- Water (e.g. ad libitum): Automatic watering system, ad libitum
- Acclimation period: 34 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Maintained between 68 and 76 degrees Fahrenheit
- Humidity (%): Maintained between 40 and 70% relative humidity
- Photoperiod (hrs dark / hrs light): Approx 12 hours light and 12 hours dark, maintained by automatic timer.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Water (reagent grade water).
- Justification for choice of solvent/vehicle: The vehicle is considered, under the conditions of the assay, to be stable for the duration of the assay.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was weighed out and on the day of dosing mixed with the vehicle to provide stock solutions such that individual animal doses (volume) did not exceed 1.0 ml / 100 grams bodyweight.


The test material and vehicle were administered by oral gavage as a single dose.

Duration of treatment / exposure:

Dosed once.

Frequency of treatment:

Dosed once.

Post exposure period:

72 hours (bone marrow samples collected 24, 48 and 72 hours after dosing).

Remarks:

Doses / Concentrations:
0.15 gram/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
0.375 gram/kg
Basis:
actual ingested

Remarks:

Doses / Concentrations:
0.75 gram/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 per sex per dose.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): Considered to have been stable for the duration of the assay since it produced the expected level of mutagenic activity.
- Route of administration: intraperitoneal injection.
- Doses / concentrations: cyclophosphamide was dosed at 40 mg/kg

Tissues and cell types examined:

Erythrocytes in bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A rangefinding study was performed using three doses, 2.0, 1.0 and 0.5 grams test material/kg body weight. One female and one male were used for each dose group.

All animals at 0.5 g/kg survived three days after being dosed. One animal at 1.0 g/kg survived three days after being dosed. Therefore, 0.75 g/kg was used as the top dose in the assay.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Animals were euthaized by CO2 asphyxiation at approximately 24, 48 and 72 hours after dosing. Animals dosed with cyclophosphamide were sacrificed at 24 hours only.
Immediately upon euthanization of the animals, both femurs were removed. The bone marrow was then removed and suspended in fetal bovine serum. After the suspension was centrifuged, the pellet was resuspended and smears prepared (two slides per animal).


DETAILS OF SLIDE PREPARATION:
Slides were stained using acridine orange.


METHOD OF ANALYSIS:
Microscopic evaluation:
Polychromatic erythrocytes (PCE) stain flourescent red/orange, normochromatic erythrocytes (NCE) are unstained or stain a dull green, and micronuclei stain bright yellow. Additional criteria for scoring micronuclei are circular appearance and a diameter between 1/20 and 1/5 of the cells diameter.



OTHER:

Evaluation criteria:

1000 polychromatic erythrocytes (PCE) from each animal were examined for micronuclei, and the ratio of PCE's to NCE's was determined for each animal by counting 1000 erythrocytes (PCE's and NCE's).

Statistics:

Statistical analysis included calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period. When the ANOVA was significant, comparisons of vehicle control to dosed group means were by Duncans Multiple Range Test.

Residuals from the ANOVA were analysed for normality by Wilk's Criterion. The residuals were normally distributed (at the 0.01 level of significance) in more than 25% of the analyses. Therefore nonparametric analyses were not performed.

Dose related response was evaluated by Jonkheere's Test for Ordered Response.

Sexes were analyzed separately.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Overt toxicity was observed at 48 hours in the 0.75 g/kg dose group in males only

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Micronucleus Assay:
Neither a statistically signficant positive response not a dose related increase in the number of micronuclei was observed at 24, 48 or 72 hours.
Overt toxicity, as measured by a statistically signficant decrease in the percentage of polychromatic erythrocytes, was observed at 48 hours in the 0.75 g/kg dose group for the males only. The percentages of polychromatic erythrocytes observed were within the range expected for healthy mice. All postive and negative (vehicle) controls responded in a manner consistent with data from previous studies.

Necropsy:
One animal died during the study (a female in the 0.375 g/kg group).
Necropsy findings: Uterus/cervix; moderately enlarged, filled with clear colourless liquid.
The exact casue of death for this animal is unknown.

Table 1 (attached background material): Data Report Summary - Contains data for the three time points by listing the means and standard deviations of the micronuclei data for each dose group and sex.

Conclusions:

Interpretation of results (migrated information): negative
The test substance did not induce a notable (either statistically or biologically significant increase in micronucleus formation in either sex of mice.
The assay contained positive (cyclophosphamide) and negative (vehicle) controls which responded in an appropriate manner. Under the conditions of the assay, the test substance does not produce clastogenic abnormalities for CD-1 mice at doses up to and including 0.75 gram/kg at the times tested.

Executive summary:

The objective of the study was to determine whether the test substance was capable of inducing micronuclei formation in mouse bone marrow.

Test concentrations were 0.15, 0.375 and 0.75 g/kg administered by gavage in single doses. Levels exceeding 0.75 g/kg were toxic. Bone marrow samples were collected and evaluated for micronucleus formation 24, 48 and 72 hours after dosing.

The test substance did not induce a statistically significant increase in micronucleus formation at any of the collection times surveyed. Thus the test substance was not clastogenic in mouse bone marrow under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

A full complement of relevant in vitro genetic toxicity studies yielded negative results for the substance (Traul, 1984; Traul, 1983; Przygoda, 1988a and Przygoda, 1988b). An additional in vivo mammalian erythrocyte micronucleus test conducted on the substance also yielded a negative result. No more testing is deemed necessary and the substance is not classified under genetic toxicity.

Justification for selection of genetic toxicity endpoint
In Vivo study used as most relevant study to confirm negative classification for genetic toxicity, with standard suite of supporting in vitro studies.

Justification for classification or non-classification

The standard suite of in vitro studies required under REACH, and an in vivo mammalian erythrocyte micronucleus test were all negative. The substance therefore does not meet the criteria for classification under genetic toxicity.