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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse mutation: Gene mutation test was performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) to determine the mutagenic nature of the test chemical. No cytotoxicity was observed till 10000 μg/plate dose range. Thus the test chemical was considered to be not mutagenic in nature.

Chromosomal Abberation test: A Gene mutation study was performed according to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) to determine the genetic nature of the test chemical. No cytotoxicty was observed  till concentration of 5000 μg/ml with and without metabollic activation system. Thus the test chemical can be considered as not mutagenic in nature.

In-vitro mammalian cell gene mutation:

The test chemical was tested for mutagenic effects in Chinese hamster ovary cells at 0 (solvent control), 1, 2.5, 5 and 10 mM with and without metabolic activation. 7,12-dimethylbenz(a) anthracene and N-ethyl-N-nitrosourea were employed as positive controls with and without metabolic activation respectively. Treatment with N-ethyl-N-nitrosourea produced a significant increase in the number of revertant colonies whereas 7,12-dimethylbenz(a) anthracene did not. As such, the performance of the metabolic activation system could not be ascertained in the experiment. Without metabolic activation, the test chemical failed to produce a significant increase in the number of revertant colonies when compared to control data. No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Not specified
Species / strain / cell type:
bacteria, other: Salmonella typhimurium, TA98, TA100, TA1535, and TA1538
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal enzymes from male Spraque-Dawley rats
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 667...10000 μg/plate
Concentration range in the main test (without metabolic activation): 667...10000 μg/plate
Vehicle / solvent:
Solvent: Acetone
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 667 μg/plate
Rationale for test conditions:
not specified
Evaluation criteria:
not specified
Statistics:
not specified
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 μg/plate
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>5000 μg/plate
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>10000 μg/plate
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>10000 μg/plate
Additional information on results:
Observations:
The vihicle control value for tester strain TA98 in the presence of microsomal enzymes was outside the acceptable range. Therefore, tester strain TA98 was retested in the presence of microsomal enzymes.
Remarks on result:
other: other: preliminary test
Conclusions:
Gene mutation test was performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) to determine the mutagenic nature of the test chemical. No cytotoxicity was observed till 10000 μg/plate dose range. Thus the test chemical was considered to be not mutagenic in nature.
Executive summary:

Gene mutation test was performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) to determine the mutagenic nature of the test chemical. Salmonella typhimurium, TA98, TA100, TA1535, and TA1538 starins were used with and without metabolic activation system  obtained from Liver microsomal enzymes from male Spraque-Dawley rats. Concentration range in the main test (with metabolic activation): 667 to 10000 μg/plate and Concentration range in the main test (without metabolic activation): 667 to 10000 μg/plate were used. No cytotoxicity was observed till 10000 μg/plate dose range. Thus the test chemical was considered to be not mutagenic in nature.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from Study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: non-bacteriological in vitro 6th amendment data
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal enzymes from male Spraque-Dawley rats
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 1250...5000 μg/ml
Concentration range in the main test (without metabolic activation): 1250...5000 μg/ml
Vehicle / solvent:
Carboxymethylcellulose sodium
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Carboxymethylcellulose sodium
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 24 hours
Exposure period (without metabolic activation): 48 hours

Fixation:
6th Amendment fixation time: 2
Rationale for test conditions:
No data
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1100 μg/ml
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>5000 μg/ml
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>5000 μg/ml
Additional information on results:
Observations: none
Remarks on result:
other: other: preliminary test
Conclusions:
A Gene mutation study was performed according to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) to determine the genetic nature of the test chemical. No cytotoxicty was observed till concentration of 5000 μg/ml with and without metabollic activation system. Thus the test chemical can be considered as not mutagenic in nature.
Executive summary:

A Gene mutation study was performed according to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) to determine the genetic nature of the test chemical. The test was performed on Chinese hamster lung fibroblasts (V79) cells with and without metabollic activation at Concentration range in the main test (with metabolic activation): 1250 to 5000 μg/ml and Concentration range in the main test (without metabolic activation): 1250 to 5000 μg/ml. The S9 metabolites were obtained from liver microsomal enzymes from male Spraque-Dawley rats. Carboxymethylcellulose sodium was used a vehicle and the study was performed for 24 hours with metabollic activation and 48 hours without metabollic activation system. No cytotoxicty was observed till concentration of 5000 μg/ml with and without metabollic activation system. Thus the test chemical can be considered as not mutagenic in nature.

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:
guideline study
Justification for type of information:
WoE study
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The purpose of this study was to assess toxic and genotoxic effects of the given test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.
This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthineguanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG. Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated
cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
6
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
7
Details on mammalian cell type (if applicable):
Cell line used: Chinese Hamster Ovary (CHO) cells
Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine suppl
emented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
6) 0, 1.0, 2.5, 5.0 or 10.0 mM
7) 0, 0.1, 0.25, 0.5 or 1 mM
Vehicle / solvent:
6)
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Test chemical was easily dissolved in water

7)
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/ vehicle: The test chemical was easily dissolved in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: N-ethyl-N-nitrosourea (ENU) (2.5 mM) without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
Details on test system and experimental conditions:
6)
METHOD OF APPLICATION: In medium with pre-incubation
DURATION- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)
SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet
NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated
DETERMINATION OF CYTOTOXICITY- Cytotoxicity test: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

7)
METHOD OF APPLICATION: In medium with pre-incubation
DURATION
- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterinthymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable
Rationale for test conditions:
No data
Evaluation criteria:
6) No data
7) The plates were scored for total number of colonies
Statistics:
6) To quantify the amount of dispersion of the different data values, calculations of the standard deviation(s) was performed when applicable.
7) No data
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not valid
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:
not valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:pH and osmolality was not determined in the gene mutation test.

RANGE-FINDING/SCREENING STUDIES (if applicable): Initial tests of dose-finding was completed without S9-induced metabolic activation. A range of test concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mM) was applied at a volume of 20 microliter per applicable well 24 hrs after seeding to single cultures (4 x 103 cells per well) in fresh medium (180 microliter per well) in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hrs after treatment using the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. In the MTT assay, 20 microliter of MTT solution was added to each well and the 96-well plates were returned to the incubator for 1 hour. After 1 hour, the MTT solution was removed by gently pouring out the fluid from the plate onto a paper cloth. Each well was washed gently with two times of 100 microliter of PBS. The blue formazan product was dissolved by addition of 100 microliter of 100% DMSO per well. The plates were swirled gently for 10 min. to dissolve the precipitate. Absorbance was monitored at 540 nm using a SpectraMax M2 Microplate Reader and the software SoftMax Pro® (Molecular Devices, Sunnyvale, CA, USA). After 24 and 48 hrs of treatment, the medium of the 96-well plates destined for analyzing protein content was removed by gently pouring out the fluid from the plate onto a paper cloth. Each well was washed gently with two times of 100 microliter of PBS before 50 microliter of Mammalian protein extraction reagent (M-PER) solution was added to each well. The plates were swirled gently overnight before 25 microliter of the cell lysate were transferred to a new 96-well plate together with 25 microliter bovine serum albumin (25 microliter of 5-750 micrgram/ml in M-PER) as a standard and with 25 microliter of M-PER as a blank. A solution, containing 50 part of BCA Reagent A (containing sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1 M sodium hydroxide) and 1 part of BCA Reagent B (containing 4% cupric sulfate), was added to each well and the plates were mixed thoroughly on a plate shaker for 30 sec. The plates were then covered and incubated for 30 min. at 37°C. After cooling the plate to RT, absorbance was measured at 562 nm using a Multiskan™ FC Microplate Photometer and the software ScanIt™ (Thermo Fisher Scientific, Waltham, MA, USA). A minimum of 6 replicates per dose concentration from 3 independent cultures was performed for each experiment. Results obtained by the two cytotoxicity assays were expressed as percentage of controls. From the basis of the results from the MTT and BCA assays, test concentrations of the chemical was chosen to be included in the gene toxicity test.
All tests of the preliminary dose-finding study was completed without S9-induced metabolic activation. The test chemical was added to the cells in the test concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mM) and the MTT and BCA assays were used to evaluate the chemical effects on cell viability and total protein concentration, respectively. Based on the results from the MTT and BCA assays, revised test concentrations of the chemical may be chosen to be included in the gene toxicity test. pH and osmolality was not determined in the preliminary dose-finding/toxicity test.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: the positive controls ENU and 7,12-dimethylbenz(a) anthracene showed indication of gene mutations occurring while no other treatment gave rise to gene toxicity.When the mutation frequency was determined, a frequency of -2.61 x 10-4 was shown after a 3 hour exposure of 7,12-dimethylbenz(a) anthracene in the presence of 4% S9 liver microsomal fraction, and 3.84 x 10-4 for ENU as the positive control and in the absence of S9 liver microsomal fraction.

Cytotoxicity test
Cells (0.5 x 105 per well) were seeded in fresh medium (2 ml per well) in 6 well-plates and were incubated overnight at 37°C, 5% CO2. On the day of exposure, the test chemical added to each applicable well to give a final concentrations of 0, 1.0, 2.5, 5.0 or 10.0 mM and in the absence or 4% (80 microliter) presence of S9-induced metabolic activation. Negative controls (cell medium), solvent/vehicle controls (PBS) and positive control substances were also included in each experiment. ENU was used as positive control substance in experiments without metabolic activation, while 7,12-dimethylbenz(a) anthracene was used in experiments with metabolic activation. During the exposure period, treated cultures were incubated at 37°C, 5% CO2. After the 3 hour exposure period, cells were washed several times with sterile PBS to remove the test chemical. Cells were then trypsinized and re-seeded in duplicates from two parallel duplicate cultures into new 6-well plates, containing fresh medium, in the concentration of 0.5 x 105 cells per well. The cultures were then incubated for 24 and 48 hrs at 37°C, 5% CO2. The relative total growth and cytotoxicity was evaluated 24 and 48 hrs after seeding by counting in a haemocytometer.
A minimum of 2 replicates per dose concentration, including negative and positive control, were performed for each experiment. The test chemical showed a slight evidence of cytotoxicity after 24 and 48 hrs of treatment with 5 mM. No results of the BCA assay are presented since we had a major technical problem with the Multiskan™ FC Microplate Photometer, leaving us with no possibilities to analyze our BCA tests for ethyltriphenylphosphonium bromide at the given time.Since a minor level of cytotoxicity was observed in the MTT assay at 5.0 mM after 24 and 48 hrs of treatment in this preliminary dose-finding test, further testing concentrations were revised and adapted to have a maximum test concentration of 10.0 mM.
Remarks on result:
other: not mutagenic
Conclusions:
The test chemical was tested for mutagenic effects in Chinese hamster ovary cells at 0 (solvent control), 1, 2.5, 5 and 10 mM with and without metabolic activation. 7,12-dimethylbenz(a) anthracene and N-ethyl-N-nitrosourea were employed as positive controls with and without metabolic activation respectively. Treatment with N-ethyl-N-nitrosourea produced a significant increase in the number of revertant colonies whereas 7,12-dimethylbenz(a) anthracene did not. As such, the performance of the metabolic activation system could not be ascertained in the experiment. Without metabolic activation, the test chemical failed to produce a significant increase in the number of revertant colonies when compared to control data. No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data.
Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells.The study was performed as per OECD 476 Guidelines. A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations of the test chemical were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)and the bicinchoninic acid(BCA) assay to assess cell viability and protein concentration, respectively.From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.Cells (0.5 x 105per well) were seeded in fresh medium (2 ml per well) in 6 well-plates and were incubated overnight at 37°C, 5% CO2. On the day of exposure, the test chemical was added to each applicable well to give a final concentrations of 0, 1.0, 2.5, 5.0 or 10.0 mM and in the absence or 4% (80ml) presence of S9-induced metabolic activation. Negative controls (cell medium), solvent/vehicle controls (PBS) and positive control substances were also included in each experiment.N-ethyl-N-nitrosourea (ENU) (2.5 mM) was used as positive control substance in experiments without metabolic activation, while 7,12-dimethylbenz(a) anthracene (8mM) was used in experiments with metabolic activation. pH and osmolality was not determined in the preliminary dose-finding/toxicity test. During the exposure period, the treated cultures were incubated at 37°C, 5% CO2.After the 3 hour exposure period, cells were washed several times with sterile PBS to remove the test chemical. The cultures were then incubated for 7 days at 37°C, 5% CO2. During the expression period, the cell medium was exchanged every second day. However, if the cell population increased so that cells covered more than 90% of the well surface, cells were sub-cultured.At the end of the expression period, cells were trypsinized and the number cells per ml and well was calculated manually using a heamocytometer. Cells were transferred to new 6 well plates with or without TG added as a selection/restrictive agent. Four 6-well plates were prepared per culture, i.e. duplicate plates for viable count evaluation and duplicate plates for TG-treatment. The plates were incubated for 14 days at 37°C in a humidified atmosphere enriched with 5% CO2. At the end of the 14 day incubation period, the cells were fixed with 100% methanol for 10 min. and then stained with a 0.5% Crystal Violet solution. The plates were scored for total number of colonies by manual counting. As a result, the mutation frequency could be calculated (see equation below).A minimum of 2 replicates per dose concentration, including negative and positive control, were performed for each experiment. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. Treatment with N-ethyl-N-nitrosourea produced a significant increase in the number of revertant colonies whereas 7,12-dimethylbenz(a) anthracene did not. As such, the performance of the metabolic activation system could not be ascertained in the experiment. Without metabolic activation, the test chemical failed to produce a significant increase in the number of revertant colonies when compared to control data. No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data. No cytotoxic effects were observed when CHO cells were exposed to the test chemical for 3 hrs.

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-Nnitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony was seen in one well out of four at the concentration of 0.5 mM and in the absence with 4% S9 liver microsomal fraction, and two very diffuse colonies were detected in one well out of four at the concentration of 0.1 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the three spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of methyl 2-napthyl ether, i.e. these concentrations did not show any evidence of diffuse or clear colonies present. When the mutation frequency was determined, a frequency of 5.35 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of methyl 2-napthyl ether in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that methyl 2-napthyl ether does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs. Based on the results of the current study, we conclude that the test chemical does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hrs, in the presence or absence of metabolic activation.

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

Additional information

Bacterial Reverse mutation:

1. Gene mutation test was performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) to determine the mutagenic nature of the test chemical. Salmonella typhimurium, TA98, TA100, TA1535, and TA1538 starins were used with and without metabolic activation system  obtained from Liver microsomal enzymes from male Spraque-Dawley rats. Concentration range in the main test (with metabolic activation): 667 to 10000 μg/plate and Concentration range in the main test (without metabolic activation): 667 to 10000 μg/plate were used. No cytotoxicity was observed till 10000 μg/plate dose range. Thus the test chemical was considered to be not mutagenic in nature.

2. Bacterial Reverse Mutation Assay was performed on S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 to determine the mutagenic nature of test chemical. The test was performed With and without metabollic activation. The dose concentrations used were 100, 333, 1000, 2500, and 5000 mg/plate. Precipitate on plates was noted at 2500 and 5000 mg/plate doses. Background lawns appeared intact. Thus the test chemical appeared to be non mutagenic in nature.

3. Bacterial reverse mutation study was performed on S. typhimurium TA 1535, TA 1537, TA 98 and TA 100; E. coli WP2 uvr starins to determine the mutagenic nature of test chemical. The study was performed in the presence and absence of mutagenic activation provided by rat liver S9 fraction at dose concentrations of 312.5 - 5000 μg/plate. No mutagenecity was observed indicating that the test chemical is non mutagenic in nature.

Chromosomal Abberation test:

A Gene mutation toxicty study was performed according to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) to determine the genetic nature of the test chemical. The test was performed on Chinese hamster lung fibroblasts (V79) cells with and without metabollic activation at Concentration range in the main test (with metabolic activation): 1250 to 5000 μg/ml and Concentration range in the main test (without metabolic activation): 1250 to 5000 μg/ml. The S9 metabolites were obtained from liver microsomal enzymes from male Spraque-Dawley rats. Carboxymethylcellulose sodium was used a vehicle and the study was performed for 24 hours with metabollic activation and 48 hours without metabollic activation system. No cytotoxicty was observed till concentration of 5000 μg/ml with and without metabollic activation system. Thus the test chemical can be considered as not mutagenic in nature.

In-vitro mammalian cell gene mutation:

Various studies have been reviewed to determine the mutagenic nature of the test chemical . These include in vivo experimental studies performed;

2) An in vitromammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells.The study was performed as per OECD 476 Guidelines. A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations of the test chemical were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)and the bicinchoninic acid(BCA) assay to assess cell viability and protein concentration, respectively.From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.Cells (0.5 x 105per well) were seeded in fresh medium (2 ml per well) in 6 well-plates and were incubated overnight at 37°C, 5% CO2. On the day of exposure, the test chemical was added to each applicable well to give a final concentrations of 0, 1.0, 2.5, 5.0 or 10.0 mM and in the absence or 4% (80ml) presence of S9-induced metabolic activation. Negative controls (cell medium), solvent/vehicle controls (PBS) and positive control substances were also included in each experiment.N-ethyl-N-nitrosourea (ENU) (2.5 mM) was used as positive control substance in experiments without metabolic activation, while 7,12-dimethylbenz(a) anthracene (8mM) was used in experiments with metabolic activation. pH and osmolality was not determined in the preliminary dose-finding/toxicity test. During the exposure period, the treated cultures were incubated at 37°C, 5% CO2.After the 3 hour exposure period, cells were washed several times with sterile PBS to remove the test chemical. The cultures were then incubated for 7 days at 37°C, 5% CO2. During the expression period, the cell medium was exchanged every second day. However, if the cell population increased so that cells covered more than 90% of the well surface, cells were sub-cultured.At the end of the expression period, cells were trypsinized and the number cells per ml and well was calculated manually using a heamocytometer. Cells were transferred to new 6 well plates with or without TG added as a selection/restrictive agent. Four 6-well plates were prepared per culture, i.e. duplicate plates for viable count evaluation and duplicate plates for TG-treatment. The plates were incubated for 14 days at 37°C in a humidified atmosphere enriched with 5% CO2. At the end of the 14 day incubation period, the cells were fixed with 100% methanol for 10 min. and then stained with a 0.5% Crystal Violet solution. The plates were scored for total number of colonies by manual counting. As a result, the mutation frequency could be calculated (see equation below).A minimum of 2 replicates per dose concentration, including negative and positive control, were performed for each experiment. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. Treatment with N-ethyl-N-nitrosourea produced a significant increase in the number of revertant colonies whereas 7,12-dimethylbenz(a) anthracene did not. As such, the performance of the metabolic activation system could not be ascertained in the experiment. Without metabolic activation, the test chemical failed to produce a significant increase in the number of revertant colonies when compared to control data. No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data. No cytotoxic effects were observed when CHO cells were exposed to the test chemical for 3 hrs.

3) An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of methyl 2-napthyl ether when administered to Chinese Hamster Ovary (CHO) cells. The test substance was administered to CHO cells for 3 hours at the dose levels of 0, 0.1, 0.25, 0.5 or 1.0 mM in the absence or presence of exogenous metabolic activation (S9 mix). CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such asN-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity.No mutant colonies were detected following the exposure to positive control of7,12-dimethylbenz(a) anthracene and, therefore no conclusion could be drawn from experiments performed in the presence of exogenous metabolic activation. No clear indication of gene mutation was seen when CHO cells were treated with methyl 2-naphthyl etherat concentration of0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hoursin the absence of S9 mix. One very diffuse colony was seen in one well out of four at the concentration 0.5 mM, but this colony was a small cluster of apoptotic cells as it was only mildly coloured by crystal violet and, consequently it was considered irrelevant tothepresenttest.This was further supported by the results of the higher tested concentrations of methyl 2-napthyl ether, i.e. these concentrations did not show any evidence of diffuse or clear colonies present.In addition, no cytotoxic effects were observed when CHO cells were exposed to the test substance at any concentration used. In conclusion,methyl 2-napthyl etherdoes not give rise to gene mutations when CHO cells are exposed to the test chemical at 0, 0.1, 0.25, 0.5 or 1.0 mM for 3 hours in the absence of exogenous metabolic activation (-S9 mix). In addition,methyl 2-napthyl etherdoes not induce cytotoxic effects at concentrations ≤ 10.0 mM. 

Justification for classification or non-classification

Gene mutation study was performed to determine the mutagenic nature of the test chemical. No mutations were observed in Bacterial Reverse mutation test, Chromosomal Abberation test and In-vitro mammalian cell gene mutation test indicating that the test chemical is non mutagenic in nature.