Oxacycloheptadec-10-en-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene Toxicity In-Vitro:

Ames Assay:

Gene toxicity in vitro study was performed to evaluate the mutagenic potential of test chemical. The test was performed using Salmonella typhimurium TA1535, TA1537, TA98, TA100, and TA102 both with and without metabolic activation system using the test material at a dose level of 33, 100, 1000, 2500, or 5000 µg/plate using ethanol as solvents. Plate incorporation protocol was followed. Test substance was considered to be non mutagenic to S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without metabolic activation in the Plate incorporation assay. Hence the test substance cannot be classified as mutagenic in vitro.

In-Vitro chromosomal aberration assay:

A chromosome aberration assay using peripheral human lymphocytes with and without metabolic activation (S9) was negative. The positive controls elicited the expected results. No clastogenic findings were observed for the test article. Thus, based on all the above observations and results , it was concluded that the test chemical did not induce any mutagenicity at any tested concentration in human lymphocytes. In another study, the test chemical was tested in vitro with human lymphocytes. The concentrations ranged from 17.81 to 2280 lg/ ml with the S9 cultures and 4.45 to 71.25 lg/ml without the S9 cultures. Since, the test chemical did not induce significant increases in the frequency of cells with chromosome aberrations or polyploidy cells in either the presence or absence of a liver enzyme metabolizing system, it was concluded that it is not clastogenic to human lymphocytes in vitro.

In vitro Mammalian cell gene mutation assay:

An in vitro mutagenicity test was performed on L5178Y mouse lymphoma cells to assess the mutagenicity of the test chemical. The assay was performed on duplicate sets both in the presence and absence of exogenous metabolic activation system. In the first set, cells were exposed to concentrations ranged from 2.5 to 40 mg/ml in the absence of metabolic activation (-S9 mix) and 5–80 mg/ml in the presence of metabolic activation (+S9). In the second experiment, concentrations employed ranged from 5-50 mg/ml (-S9) and 10-70 mg/ml (+S9). Positive and concurrent vehicle controls were also included in the assay. As seen by the results, the test substance did not induce mutation in L5178Y mouse lymphoma cell at any concentration tested, neither in the presence nor in the absence of metabolic activation and, consequently it was considered non-mutagenic (negative) in mouse lymphoma cell line, under the described experimental conditions.

Link to relevant study records

Reference

Reference 1

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Bacterial gene mutation test was performed to evaluate the mutagenic potential of test substance using both plate incorporation and preincubation protocols.

GLP compliance:

no

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

33, 100, 1000, 2500, or 5000 µg/plate

Vehicle / solvent:

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: Plate incorporation and preincubation protocols

DURATION
- Preincubation period: No data available
- Exposure duration: No data available
- Expression time (cells in growth medium): No data available
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available

Rationale for test conditions:

No data

Evaluation criteria:

Increase in revertants/plate was observed

Statistics:

No data available

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data available

Remarks on result:

other: No mutagenic potential

Conclusions:

Test substance did not produce reverse mutation up to 5000 µg/plate in Salmonella typhimurium tester strains of TA 1535, TA 1537, TA 98, TA 100 and TA 102 both with and without metabolic activation.

Executive summary:

Bacterial reverse mutation assaywas performed to evaluate the mutagenic potential of test chemicalin Salmonella typhimurium tester strains of TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed employing both plate incorporation and preincubation protocols.The test chemical was dissolved in ethanol and tested in concentrations of 33, 100, 1000, 2500, or 5000 µg/plate with and without rat liver microsomal activation (S9 mix). No substantial increase in number of mutant colonies compared to vehicle control was observed up to the concentration of 5000µg/plate. Hence,the test chemicalwas regarded non-mutagenic (negative) in bacterial reverse mutation assay under the experimental conditions described.  

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Weight of evidence approach based on the available data of the read-across chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

Equivalent or similar to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No Data Available

Species / strain / cell type:

lymphocytes:

Remarks:

Study 2 and 3: Human Lymphocytes

Cytokinesis block (if used):

Study 3: Mitosis was arrested by the addition of demecolcine (Colcemid, 0.1 ug/ml) 2 h before the harvest time.

Metabolic activation:

with and without

Metabolic activation system:

Study 2: A chromosome aberration assay using peripheral human lymphocytes with and without metabolic activation (S9) was negative.
Study 3: The concentrations ranged from 17.81 to 2280 ug/ ml with the S9 cultures and 4.45 to 71.25 ug/ml without the S9 cultures.

Test concentrations with justification for top dose:

No Data Available

Vehicle / solvent:

No Data Available

Untreated negative controls:

other:

Remarks:

Study 2: yes

Negative solvent / vehicle controls:

other:

Remarks:

Study 2: not specified

True negative controls:

other:

Remarks:

Study 2: not specified

Positive controls:

other:

Remarks:

Study 2: yes;

Positive control substance:

other: Please see 'remarks'

Remarks:

Study 3: Positive controls were ethyl methanesulfonate (500 ug/ml) in dimethylsulfoxide when S9 was absent and cyclophosphamide (25 ug/ml) dissolved in the culture medium without serum for cultures where S9 was included.

Details on test system and experimental conditions:

Study 2: A chromosome aberration assay using peripheral human lymphocytes with and without metabolic activation (S9) was negative.
Study 3: The concentrations ranged from 17.81 to 2280 ug/ ml with the S9 cultures and 4.45 to 71.25 ug/ml without the S9 cultures. Chromosome damage was assessed by counting the first 100 consecutive ‘‘well-spread’’ metaphases from each culture. Gaps, breaks, or rearrangements were noted. Cells with more than 44 chromosomes were also noted. The mitotic index was based on a count of 2000 lymphocytes, noting those in metaphase.

Rationale for test conditions:

No Data Available

Evaluation criteria:

No Data Available

Statistics:

No Data Available

Species / strain:

lymphocytes:

Remarks:

Study 2 and 3: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The chemicals in Study 2 and Study 3 were found to be non-genotoxic.

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Remarks:

Study 3: The vehicle control gave frequencies within historical control values.

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Study 3: Both positive controls gave statistically significant increases in the frequency of cells with aberrations.

Additional information on results:

Study 2: In experiment 1, doses of 18.59, 37.19, 74.38, 148.75, 297.5, 595, 1190, or 2380 ug/ml (±S9) were exposed up to 20 h. In experiment 2, the cells were dosed up to 600 ug/ml (+S9) and 150 ug/ml (S9) then exposed for 20 h or dosed up to 300 ug/ml (+S9) and 150 ug/ ml (S9) then exposed for 44 h. The positive controls elicited the expected results. No clastogenic findings were observed for the test article.

Study 3: The test chemical was not found to be mutagenic at any given dose concentration,

Remarks on result:

other: Study 2 and 3: No mutagenic potential of the test chemical was observed.

Conclusions:

Based on all the above observations and results, it has been concluded that the test chemical is not clastogenic as it has not induced chromosome aberration at any dose tested in human lymphocytes neither in presence nor in the absence of metabolic activation system.

Executive summary:

In vitro chromosomal aberration studies:

The clastogenic nature of the test chemical was assessed by in vitro mammalian chromosomal aberration test using cultured human lymphocytes, as test system.

Study 1:

The clastogenic activity of the test compound was assessed in a chromosome aberration assay using peripheral human lymphocytes. Duplicate cultures were treated with the test chemical at graded concentrations and two exposure periods (20 and 44 hours). In experiment 1, cells were exposed to doses of 18.59, 37.19, 74.38, 148.75, 297.5, 595, 1190, or 2380 mg/ml up to 20 hours both in the presence and in the absence of metabolic activation system (S9 mix). In experiment 2, the cells were dosed with 37.5, 75, 150, 225, 300, 600 (+S9 mix) or with 18.75, 37.5, 75, 112.5, 150 (-S9 mix) then exposed for 20 hours. Alternatively, cells were exposed to doses of 150, 225, 300 (+S9 mix) or 37.5, 75, 112.5, 150 (-S9 mix) then exposed for 44 hours. The positive controls elicited the expected results. No clastogenic findings were observed for the test compound at any doses applied, neither in the presence nor in the absence of metabolic activation, in both experiments. Hence, the test chemical was considered non-clastogenic (negative) in a chromosome aberration test, in peripheral human lymphocytes under the described experimental conditions.

Study2:

A chromosome aberration assay was conducted using human lymphocytes to assess the clastogenic nature of the test substance. The test chemical was employed in concentrations ranged from 17.81 to 2280 ug/ml with metabolic activation (+S9) and from 4.45 to 71.25 ug/ml without metabolic activation (-S9) for 20 hours. Positive and concurrent solvent control were also included in the text. Chromosome damage was assessed by counting the first 100 consecutive ‘‘well-spread’’ metaphases from each culture. Gaps, breaks, or rearrangements were noted. Cells with more than 44 chromosomes were also noted. Both positive controls gave statistically significant increases in the frequency of cells with aberrations, the vehicle control gave frequencies within historical control values. The test chemical did not induce significant increase the frequency of cells with chromosome aberrations or polyploidy cells neither in the presence or nor in the absence of a liver enzyme metabolizing system. Hence, it was concluded that the test chemical is not clastogenic to human lymphocytes in vitro, under the described experimental conditions.

Based on all the above observations and results, ithas beenconcluded that the test chemicalis notclastogenic as it has not induced structural or numerical chromosome aberrationat anydose testedin human lymphocytes neither in presence nor in the absence of metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data from handbook or collection of data

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Weight of evidence prepared from various studies mention below
Study 5: The gene mutation study was conducted according to L5178Y TK+/- Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical
Study 6: To evaluate the mutagenic potential of test substance in Chinese hamster ovary (CHO) K1 cells by HGPRT Assay.

GLP compliance:

not specified

Type of assay:

other: Study 5: in vitro mammalian cell gene mutation tests using the thymidine kinase gene. Study 6: HGPRT Assay

Target gene:

Study 5: Thymidine kinase
Study 6: HGPRT

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK ±3.7.2c mouse lymphoma cells were used in this study.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

) K1 cells

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No Data Available

Metabolic activation:

with and without

Metabolic activation system:

Study 5: In the first set, concentrations ranged from 2.5 to 40 ug/ml in the absence of metabolic activation (S9) and 5–80 ug/ml in the presence of metabolic activation (+S9). In the second set, concentrations ranged from 5–50 ug/ml without S9 metabolic system and 10–70 ug/ml with S9 metabolic system.

Test concentrations with justification for top dose:

Study 5: 1st set: In absence of metabolic activation system: 40 ug/ml and in presence of metabolic activation system: 80 ug/ml.
2nd set: In absence of metabolic activation system: 50 ug/ml and in presence of metabolic activation system: 70 ug/ml.
Study 6: 8 – 80 µg/mL

Vehicle / solvent:

No Data Available

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Details on test system and experimental conditions:

Study 5: In the first set, concentrations ranged from 2.5 to 40 ug/ml in the absence of metabolic activation (S9) and 5–80 ug/ml in the presence of metabolic activation (+S9). In the second set, concentrations ranged from 5–50 ug/ml without S9 metabolic system and 10–70 ug/ml with S9 metabolic system.

Rationale for test conditions:

No data

Evaluation criteria:

Study 6: Mammalian cells were evaluated for mutagenic potential.

Statistics:

No data available

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

There were no effects with the test chemical.

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

Positive controls elicited the expected results

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

K1 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenicity potential of the test chemical was determined at the all the concentration tested.

Conclusions:

Based on all the above observations and results, it has been concluded that the test chemical is not mutagenic at doses tested both in the presence and in the absence of metabolic activation.

Executive summary:

In vitro mammalian cell gene mutation assays:

Study 7:

An in vitro mutagenicity test was performed on L5178Y mouse lymphoma cells to assess the mutagenicity of the test chemical. The assay was performed on duplicate sets both in the presence and absence of exogenous metabolic activation system. In the first set, cells were exposed to concentrations ranged from 2.5 to 40 mg/ml in the absence of metabolic activation (-S9 mix) and 5–80 mg/ml in the presence of metabolic activation (+S9). In the second experiment, concentrations employed ranged from 5-50 mg/ml (-S9) and 10-70 mg/ml (+S9). Positive and concurrent vehicle controls were also included in the assay. As seen by the results, the test substance did not induce mutation in L5178Y mouse lymphoma cell at any concentration tested, neither in the presence nor in the absence of metabolic activation and, consequently it was considered non-mutagenic (negative) in mouse lymphoma cell line, under the described experimental conditions.

Study 8:

An in vitro mammalian cell gene mutagenicity test using the Hprt gene was performed using Chinese hamster ovary K1 cells to investigate the mutagenic potential of the test chemical. The cells were exposed to the test chemical at concentrations of 0 (vehicle control), 20, 40, 60, 80 µg/ml for 4 hours. The test was conducted on duplicates both in the presence and absence of exogenous metabolic activation (S9 mix). Positive and concurrent negative controls were also included in the assay. Statistically significant and dose-related increase in mutant frequency over the control was considered as positive result. No cytotoxicity was observed up to 80 µg/ml. In addition, the test substance did not increase the mutant frequency at any doses tested, neither in the presence nor in the absence of metabolic activation. Hence, the test chemical was not mutagenic at Hprt locus in CHO cells both with and without metabolic activation, in a mammalian gene mutation test.

Based on all the above observations and results, ithas beenconcluded that the test chemicalhas notinducedgene mutationat any tested concentrationneither in thepresencenor in theabsence of S9 metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data fonthe test chemicalsand read across analogueswerereviewed to determine thegenotoxicnature of the test chemical. The studies are as mentioned below:

 

AMES test:

#1

Bacterial reverse mutation assay was performed to evaluate the mutagenic potential of test chemical in Salmonella typhimurium tester strains of TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed employing both plate incorporation and preincubation protocols. The test chemical was dissolved in ethanol and tested in concentrations of 33, 100, 1000, 2500, or 5000 µg/plate with and without rat liver microsomal activation (S9 mix). No substantial increase in number of mutant colonies compared to vehicle control was observed up to the concentration of 5000µg/plate. Hence, the test chemical was regarded non-mutagenic (negative) in bacterial reverse mutation assay under the experimental conditions described.  

In vitro chromosomal aberration study:

#1

The clastogenic activity of the test compound was assessed in a chromosome aberration assay using peripheral human lymphocytes. Duplicate cultures were treated with the test chemical at graded concentrations and two exposure periods (20 and 44 hours). In experiment 1, cells were exposed to doses of 18.59, 37.19, 74.38, 148.75, 297.5, 595, 1190, or 2380 mg/ml up to 20 hours both in the presence and in the absence of metabolic activation system (S9 mix). In experiment 2, the cells were dosed with 37.5, 75, 150, 225, 300, 600 (+S9 mix) or with 18.75, 37.5, 75, 112.5, 150 (-S9 mix) then exposed for 20 hours. Alternatively, cells were exposed to doses of 150, 225, 300 (+S9 mix) or 37.5, 75, 112.5, 150 (-S9 mix) then exposed for 44 hours. The positive controls elicited the expected results. No clastogenic findings were observed for the test compound at any doses applied, neither in the presence nor in the absence of metabolic activation, in both experiments. Hence, the test chemical was considered non-clastogenic (negative) in a chromosome aberration test, in peripheral human lymphocytes under the described experimental conditions.

#2

A chromosome aberration assay was conducted using human lymphocytes to assess the clastogenic nature of the test substance. The test chemical was employed in concentrations ranged from 17.81 to 2280 ug/ml with metabolic activation (+S9) and from 4.45 to 71.25 ug/ml without metabolic activation (-S9) for 20 hours. Positive and concurrent solvent control were also included in the text. Chromosome damage was assessed by counting the first 100 consecutive ‘‘well-spread’’ metaphases from each culture. Gaps, breaks, or rearrangements were noted. Cells with more than 44 chromosomes were also noted. Both positive controls gave statistically significant increases in the frequency of cells with aberrations, the vehicle control gave frequencies within historical control values. The test chemical did not induce significant increase the frequency of cells with chromosome aberrations or polyploidy cells neither in the presence or nor in the absence of a liver enzyme metabolizing system. Hence, it was concluded that the test chemical is not clastogenic to human lymphocytes in vitro, under the described experimental conditions.

 

In vitro mammalian cell gene mutation assay:

#1

An in vitro mutagenicity test was performed on L5178Y mouse lymphoma cells to assess the mutagenicity of the test chemical. The assay was performed on duplicate sets both in the presence and absence of exogenous metabolic activation system. In the first set, cells were exposed to concentrations ranged from 2.5 to 40 mg/ml in the absence of metabolic activation (-S9 mix) and 5–80 mg/ml in the presence of metabolic activation (+S9). In the second experiment, concentrations employed ranged from 5-50 mg/ml (-S9) and 10-70 mg/ml (+S9). Positive and concurrent vehicle controls were also included in the assay. As seen by the results, the test substance did not induce mutation in L5178Y mouse lymphoma cell at any concentration tested, neither in the presence nor in the absence of metabolic activation and, consequently it was considered non-mutagenic (negative) in mouse lymphoma cell line, under the described experimental conditions.

 

#2

An in vitro mammalian cell gene mutagenicity test using the Hprt gene was performed using Chinese hamster ovary K1 cells to investigate the mutagenic potential of the test chemical. The cells were exposed to the test chemical at concentrations of 0 (vehicle control), 20, 40, 60, 80 µg/ml for 4 hours. The test was conducted on duplicates both in the presence and absence of exogenous metabolic activation (S9 mix). Positive and concurrent negative controls were also included in the assay. Statistically significant and dose-related increase in mutant frequency over the control was considered as positive result. No cytotoxicity was observed up to 80 µg/ml. In addition, the test substance did not increase the mutant frequency at any doses tested, neither in the presence nor in the absence of metabolic activation. Hence, the test chemical was not mutagenic at Hprt locus in CHO cells both with and without metabolic activation, in a mammalian gene mutation test.

Justification for classification or non-classification

The mutagenic potential ofthe target chemicalhas been thoughtfully investigated by a combination of in vitro tests: bacterial reverse mutation assay, chromosome aberration and gene mutation tests of cultured mammalian cell lines. The combination of these test approaches covers gene and chromosome mutations and clastogenic activity in somatic cells.Conclusive evidence from the studies discuss below have indicated, thatthe target chemicaldid not induce alterations in genetic material such as gene and chromosome mutations, structural and numerical chromosome aberrations in somatic cells, in vitro and, consequentlyithas beennotclassifiedfor mutagenicityaccording to the criteria of the CLP regulations.