Lithium bis[ethanedioato(2-)-κO1,κO2]difluorophosphate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three studies are available.

The bacterial reverse mutation test was performed in accordance with OECD Guideline 471. The mutagenic activity of the test substance was considered negative.

The chromosome aberration study was performed in accordance with OECD Guideline 473. The test substance was considered to cause a negative response.

The vitro mammalian cell gene mutation test was performed in accordance with OECD Guideline 490. The test item is non-mutagenic to the cultured L5178Ymouse lymphoma cells under the conditions of this study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2007-09-06 to 2007-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: 070409
Purity: 98%

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 fraction prepared from rat liver homogenates that had been treated with phenobarbital and 5,6-benzoflavone to induce drug-metabolizing enzymes was used.
- method of preparation of S9 mix : The S9Mix was prepared at a concentration of 10 v/v% by adding thawed S9 fraction that stored at 80 ℃ to the cofactor solution. The S9Mix prepared was then stored in a refrigerator until use. The composition of S9Mix per ml was as follows :
S9 fraction: 10 v/v%; MgCl2: 8 μmol; KCl: 33 μmol; G-6-P: 5 μmol; NADPH: 4 μmol; NADH: 4 μmol; sodium phosphate buffer (pH7.4): 100 μmol

Test concentrations with justification for top dose:

Range-finding study: 5, 20, 78, 313, 1250 and 5000 µg/plate (with and without metabolic activation)
Definitive study: 156, 313, 625, 1250, 2500 and 5000 µg/plate (with and without metabolic activation).

Vehicle / solvent:

- solvent(s) used: Dimethylsulfoxide
- Justification for choice of solvent: The test substance is unstable in water, but stable in dimethylsulfoxide and solubility in dimethylsulfoxide of the test substance is 10% or more. For this reason, dimethylsulfoxide was selected as the solvent.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive control substance:

other: dimethylsulfoxide

Remarks:

negative control for all bacterial stains in the presence or absence of S9Mix

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

positive control for TA 100, TA 98, WP2 uvrA in the absence of S9Mix

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

positive control for TA 1535 in the absence of S9Mix

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

positive control for TA 1537 in the absence of S9Mix

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

positive control for TA 100, TA 98, TA 1537 in the presence of S9Mix

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

positive control for TA 1535, WP2 uvrA in the presence of S9Mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in pre-incubation

DURATION
- Preincubation period: 48 hours
- Cell density at seeding (if applicable): bacterial concentrations of the growing culture in TA 100, TA 1535, WP2 uvrA, TA 98, TA 1537 were respectively 4.3, 3.1, 5.3, 3.4, 2.9 (× 10^9 cells per ml) in the preliminary test and 4.2, 3.0, 5.5, 3.4, 2.9 (× 10^9 cells per ml) in the main test.

OTHER:
- The measuring of the number of the revertant colonies: colony analyzer
- Toxic effect of the test substance: stereoscopic microscope
- Precipitation of the test substance: observed by naked eyes

Evaluation criteria:

- Acceptance criteria for the values of the negative control and the positive control:
The number of the revertant colonies of the negative control and positive control compared with the values of the historical data
- Acceptance criteria for the test result:
the growth of the contaminant in the test materials are not detected from the result of the sterility test.; the negative control values satisfy acceptance criteria; the positive control values satisfy acceptance criteria, and the number of the revertant colonies of the positive control increases two-fold or more compared with the negative control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The values of the negative controls and the positive controls were appropriate in comparison with the historical data of our laboratory. Furthermore, all of the positive controls, such as 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, benzo[α]pyrene and 2-aminoanthracene, increased the number of the revertant colonies two-fold or more compared with the negative control in all bacterial strains, respectively.

Ames test:
- Results: Neither clear dose-related increase in the number of the revertant colonies nor two-fold or more increase in the number of the revertant colonies compared with the negative control was observed in any bacterial strains at any dose levels regardless of the presence or the absence of metabolic activation. The growth of the contaminant was not observed in a result of the sterility test.
The test substance showed toxic effect in all bacterial strains in both the presence and the absence of metabolic activation at the dose of 5000 μg/plate. The test substance produced the precipitation at the dose of 5000 μg/plate of the presence of metabolic activation.

Conclusions:

The mutagenic activity of the test substance is considered negative under the test conditions employed.

Executive summary:

The mutagenic activity of the test substance was examined in the reverse mutation test by using bacterial strains according to OECD Guideline 471.

The reverse mutation test was composed of the preliminary and the main test, and performed in pre-incubation methods in all bacterial strains in both the presence and the absence of metabolic activation. After the incubation at 37 °C for 48 hours, number of revertant colonies in each bacterial strain at all dose levels was counted and compared with the negative control.

 

Neither clear dose-related increase in the number of the revertant colonies nor two-fold or more increase in the number of the revertant colonies compared with the negative control was observed in any bacterial strains at any dose levels regardless of the presence or the absence of metabolic activation. And the reappearance of the test results between the preliminary and the main test was confirmed. The values of the negative controls and the positive controls were appropriate in comparison with the historical data of our laboratory. Furthermore, all of the positive controls, such as 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, benzo[α]pyrene and 2-aminoanthracene, increased the number of the revertant colonies two-fold or more compared with the negative control in all bacterial strains, respectively.

 

It is concluded that the mutagenic activity of the test substance is considered negative under the test conditions employed.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2009-02-24 to 2009-04-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch No.: 08111121
Purity: 98%

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Cell cycle length: 13-14 h
- Sex, age and number of blood donors if applicable: two healthy, non-smoking male donors
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable: Aliquots of the cell suspension were placed in sterile universal containers and incubated at 37°C in a 5% CO2 atmosphere for approximately 48 hours. The cultures were gently shaken daily to resuspend the cells.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 5% CO2 atmosphere

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 fraction was obtained from male Sprague-Dawley derived rats, dosed with phenobarbital and 5, 6-benzoflavone to stimulate mixed-function oxidases in the liver. The S9 fraction was purchased from a commercial source and stored at -80°C or below.
- S9 mix contained: S9 fraction (10% v/v : test 1 and 25% v/v : test 2), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilised before use.

Test concentrations with justification for top dose:

First test: 10.08, 16.8, 27.99, 46.66, 77.76, 129.6, 216, 360, 600 and 1000 µg/mL.
Second test: 10.08, 16.8, 27.99, 46.66, 77.76, 129.6, 216, 360, 600 and 1000 µg/mL
The maximum concentration tested was based on the highest concentration producing a pH change of a difference of approximately 1 unit of the control.

Vehicle / solvent:

- Solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent: Prior to commencing testing, the solubility of the test substance in a solvent compatible with the test system was assessed. The test substance was found to be soluble in Dimethyl Sulphoxide (DMSO) at 252 mg/mL (1M).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive control substance:

other: Dimethyl Suphoxide (DMSO)

Remarks:

in the absence and presence of S9 mix

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

in the absence of S9 mix

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

in the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours in the first test. In the second test, 3-hour exposure was used in the presence of S9 mix; a 21-hour continuous exposure was used in the absence of S9 mix.
- Expression time (cells in growth medium): 18 hours (except in the second test in the absence of S9 mix, which used continuous exposure without a dedicated expression period)
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours (19 hours exposure and / or expression time + 2 hours incubation)

NUMBER OF REPLICATIONS: 2 cultures per concentration in each test.

NUMBER OF CELLS EVALUATED: 100 metaphase figures per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

The assay was considered to be acceptable if the negative and positive control values lay within the recent historical control range.

The test substance was considered to cause a positive response if the following conditions were met:
Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) were observed at one or more test concentration.
The increases exceed the negative control range of the laboratory, taken at the 99% confidence limit.
The increases were reproducible between replicate cultures.
The increases were not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a dose-relationship was considered to support the conclusion.

A negative response was claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies were observed, at any concentration.

Statistics:

The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).

Key result

Species / strain:

lymphocytes: Human lymphocytes in whole blood culture

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
All mean values for the vehicle control (DMSO), and all the test substance treatment concentrations were within the historical control range for this laboratory, when taken at the 99% confidence limit.
Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Genotoxicity results
- Toxicity data: In both the absence and the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control, in either test.
- Use of silanised glassware: No significance difference in cytotoxicity or chromosomal aberrations were seen between the two types of glassware.
- Polyploid analysis: No statistically significant increases in polyploid metaphases were observed during metaphase analysis in either test.

Conclusions:

The test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Executive summary:

A study was performed to assess the ability of the test substance to induce chromosomal aberrations in human lymphocytes cultured in vitro according to OECD Guideline 473.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Solvent and positive control cultures were also included. The study comprised two independent tests. In the first test, a three hour treatment was used in both the absence and presence of S9 mix. In the second test, a 21 hour continuous treatment was used in the absence of S9 mix, and a three hour treatment using an increased S9 concentration (5% v/v) was used in the presence of S9 mix. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

 

In both the absence and presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test.

Silanised glassware was used for the treatment period of all tests. Glassware that had not undergone the silanisation treatment was used to provide a comparison. No significance difference in cytotoxicity or chromosomal aberrations were seen between the two types of glassware.

All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

 

It is included that the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-06-17 to 2019-07-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Batch No.: 180727
Purity: 99.0%

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: This cell line is the sensitive cell of the mouse lymphoma assay
- Suitability of cells: This cell line is accredited by “OECD Guideline for the Testing of Chemicals No.490 (July 29, 2016)”.

For cell lines:
- Absence of Mycoplasma contamination: The absence of Mycoplasma contamination in the cells was affirmed and cell cultures were cleaned for pre-existing mutant cells.
- Number of passages if applicable: The cell passage used in this study is 15.
- Methods for maintenance in cell culture: at 35.2~37.8 ºC in the humidified atmosphere of 4.9~5.0% CO2.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 is prepared from the livers of the rats induced with Aroclor1254, and the details are shown below: male Sprague Dawley rats are treated by a single intraperitoneal injection of Aroclor 1254 at the dose of 500 mg/kg five days prior to the S9 preparation. The livers of the rats are taken out under asepsis conditions, homogenized in a 0.15 mol/L KCl solution (1g liver: 3ml KCl solution) and centrifuged at 11000rpm for 10 min with AllegraTM64R Auto Freeze Centrifuge. The supernatant of S9 is stored in liquid nitrogen.
- Preparation of 10% S9 mixture (S9 mix): 10% S9mix was prepared according to the recipe below immediately before being used and placed in the mixture of water and ice. The remaining S9mix was discarded.
Phosphate buffer (0.2 mol/L): 5.0 ml; MgCl2 (0.4 mol/L)-KCl (1.65 mol/L) solution: 0.2 ml; G-6-P-Na2 solution (140 mg/ml): 0.1 mL; NADP-Na2 solution (300 mg/mL): 0.1 mL; S9: 1.0 mL; Sterile distilled water: 3.6 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Before being used or the stored time is longer than one year, the S9 is tested for its sterility, its protein content (not higher than 40mg/ml), and its capability to activate known mutagens in the Ames test. The S9 used in this test was prepared on March12, 2018. The results of the test indicated that each index of S9 was qualified, which met the test requirements.

Test concentrations with justification for top dose:

According to the results of the preliminary test, the L5178Y cells were treated at six exposure concentrations including 1000, 400, 160, 64, 25.6 and 10.24 μg/ml in the treatment conditions of exposure for 3 hours with and without metabolic activation respectively. The cells were treated at six exposure concentrations including 400, 160, 64, 25.6, 10.24 and 4.096 μg/ml in the treatment conditions of exposure for 24 hours without metabolic activation.

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: Cyclophosphamide monohydrate (CP)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 3×10^5 cells/mL (all initial cell cultures)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: After the treatment, the cell cultures treated for 3h with and without S9 were centrifuged at 1000rpm for 5 min, washed twice with Hank’s buffer salt solution, re-suspended in 20ml RPMI10 and cultured for approx. 22 hours. The cell cultures treated for 24h without S9 were subjected to the same operations as above and counted after re-suspension in 20ml RPMI10. The resulting cell suspensions were adjusted to 3×105 cells/ml and cultured for another approx.24 hours.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): The cells were cultured for 2 days to allow mutant expression. On day1 of the expression period, the cell density was determined and diluted to 3×105 cells/ml with RPMI10. Cell cultures with less than 3×105 cells/ml were incubated without adjusting. On day2 of the expression period, the cells were counted again.
- Selection time (if incubation with a selective agent): After expression period, cells were suspended in medium with and without the selective agent triflurothymidine (TFT) for the determination of the numbers of mutants (selection plates = TFT plates) and for cloning/planting efficiency (viability plates = PE plates), respectively.
According to the cell density on day2, the cells were adjusted to 1×104 cells/ml in a volume of 50ml, called cell culture B. Then part of cell culture B was diluted to 8 cells/ml in 25ml, called cell culture D. The selective agent, trifluorothymidine (TFT), was added to the remaining cells of the culture B. The final concentration of TFT in cell culture was 3μg/ml.
For each cell culture, two 96-microwell plates were prepared with cell culture B as TFT plates, and one 96-microwell plate was prepared with cell culture D as PE plate (PE = plating/cloning efficiency). For all plates, 0.2ml of the cell culture was added into each well, which would nominally result in 2000 or 1.6 cells per well, respectively.
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used: yes, trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: The plates of 3 hours treatment group with and without S9-mix were performed on the 12 day, and the 24h treatment group was counted on 11 day. For the PE plate, empty wells and total wells were counted. For the TFT plate, wells with viable colony and total wells were counted. The number of empty wells resulted from the difference between total and colony-containing wells. In addition, for the TFT plates of the highest concentration, the solvent and positive controls, large and small colonies were counted, respectively.
- Criteria for small (slow growing) and large (fast growing) colonies:
- Large colony: Size: the diameter was not less than a quarter of diameter of the well; Morphology: thin and dispersive
- Small colony: Size: the diameter was less than a quarter of diameter of the well; Morphology: compact and nubbly

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative total growth (RTG); other: Plating efficiency (PE); mutant frequency (MF); induced mutant frequency (IMF); induced mutant frequency of small clones (IMFsc)

Evaluation criteria:

When IMF(s) at one or several dose levels are more than the GEF* of 126×10^-6, and the increase is concentration-related and/or replicated, the result is evaluated as positive.
The result is evaluated as negative, if none of the above criteria is met.
In case the criteria above are not met at the same time, the repeated test should be performed using modified experimental conditions, and the biological relevance of the result should be considered. If the result is still not clear, the result is concluded to be equivocal.

*GEF = Global Evaluation Factor, established for the MLA making use of the version of microtiter plates as average background mutation frequency of negative controls.

Statistics:

- Criteria of Solvent Control
The solvent control is considered to be valid if the groups meet all of the condition below:
Plating Efficiency (PE): 65～120%
Mutant Frequency (MF): (50~170) ×10-6
Suspension Growth (SG) (3h treatment): 8~32
Suspension Growth (SG) (24h treatment): 32~180
- Criteria of Positive Control
The positive control is considered to be valid if the groups meet all of the condition below
1) RTG>10%;
2) IMF>300×10^-6 and IMFsc>40%IMF at the same time; Or IMFsc>150×10^–6.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable): The preliminary tests for in vitro mammalian cell gene mutation test using the thymidine kinase gene has been performed twice in this lab.The results in the preliminary tests showed that no precipitation of test item was observed in all the doses. The cell count results showed that the test item produce dose-related cytotoxicity.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : In this study, the results of the solvent and positive controls met all criteria, so the sensitivity of the assay and the efficacy of the S9 mix could be validated.

For all test methods and criteria for data analysis and interpretation:
- Any other criteria: GEF for MLA

Gene mutation tests in mammalian cells:
Results: In the cell cultures of the definite test series, no test item precipitation was observed at all test concentrations, either at the beginning or the end of treatment. The results of mutant frequency showed that the IMF of all cultures exposed for 3h and 24h in the absence and present of S9 was less than 126×10^-6.

Conclusions:

The results of this study are negative, so it is concluded that the test item is non-mutagenic to the cultured L5178Ymouse lymphoma cells under the conditions of this study.

Executive summary:

The study was performed to evaluate the test item for its ability to cause gene mutations in vitro in cultured mammalian cells according to OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene).

The mouse lymphoma L5178Y (TK+/--3.7.2C) cells were exposed to six exposure concentrations including 1000, 400, 160, 64, 25.6 and 10.24 μg/mL in the treatment conditions of exposure for 3hours with and without metabolic activation respectively. The cells were treated at six exposure concentrations including 400, 160, 64, 25.6, 10.24 and 4.096 μg/mL in the treatment conditions of exposure for 24 hours without metabolic activation. The positive and solvent (DMSO) controls were included the same time.

 

In this test, the results of the solvent and positive controls met all quality criteria, so the sensitivity of the assay and the efficacy of the S9 mix could be validated.

No test item precipitation was observed at all test concentrations, either at the beginning or the end of treatment.

The results of mutant frequency showed that the induced mutant frequencies (IMF) of all cultures exposed for 3 h and 24 h were consistently less than the average background mutant frequency of 126×10^–6.

 

The results of this study are negative, so it is concluded that the test item is non-mutagenic to the cultured L5178Ymouse lymphoma cells under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

One in the mammalian erythrocyte micronucleus test was performed in accordance with OECD Guideline 474. The test substance was considered to cause a negative response.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2015-03-04 to 2015-08-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

no

Remarks:

non GLP, but in compliance with China National Metrology Accreditation

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Batch No.: 141104
Purity: 95.4%

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zhejiang Center of Laboratory Animals, Hangzhou, Zhejiang
- Weight at study initiation: 25 - 30 g
- Assigned to test groups randomly: Yes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3 °C
- Humidity (%): 40 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours light /dark cycle
- Air changes (per hr): 10 - 12

Route of administration:

other: negative control group and the nominal treatmeht groups: gavage; positive controI group: intraperitoneaI injection

Vehicle:

- Vehicle used: Vegetable oil
- Concentration of test material in vehicle: 25, 50, 100 mg/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions of the test substance in vegetable oil were prepared

Duration of treatment / exposure:

All mice were administered again 24h after the first dosing.

Frequency of treatment:

twice, with an interval 24 h

Dose / conc.:

25 other: mg/kg

Dose / conc.:

50 other: mg/kg

Dose / conc.:

100 other: mg/kg

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral gavage
- Doses / concentrations: 40 mg/kg (dissolved in physiologic solution)

Tissues and cell types examined:

Bone marrow from sternum

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
For the micronucleus assay, sternum was removed and marrow was aspirated from the bone and transferred to glass slides containing a drop of fetal calf serum. After the slides were allowed to air-dry, they were fixed in absolute methanol for 15 min, air-dried again. The bone marrow samples were stained with Giemsa solution. Briefly, Giemsa was prepared in a 1:9 solution with phosphate-buffered saline (PH 6.8), and slides were stained for approximately 20 min. The slides were then rinsed with phosphate-buffered saline (PH 6.8).

METHOD OF ANALYSIS:
Under a microscope, at least 2000 polychromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei. The frequency of micronucleated polychromatic erythrocytes (MNPCE) per 200 cells was calculated, and the ratio PCE/NCE was determined by counting a total of 200 erythrocytes. PCEs with more than one micronucleus were scored as a single MNPCE. The frequency of micronuclei in polychromatic cells provided an index of induced genetic damage.

Statistics:

The frequency of micronucleated PCE (MNPCE/200) scores was transformed by the Square Root Transformation approach, and the normality of the distribution of the transformed date was assessed by means of the method of moments. The control and treated groups were then compared by using the Dunnett’s – test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Clinical Signs / Mortality:
No clinical signs were observed in any test animal which could be suspected to be due to the test substance. And no death occurred in any test animal.

Micronucleus rate/ PCE/NCE ratio:
A statistically significant increase in the incidence of MNPCE and decrease in the polychromatic erythrocyte fraction over the control value was observed following treatment with the positive control substance cyclophosphamide ( P < 0.01), relative to the negative control in either male or female mouse bone marrow. The values for three treated doses for males and females did not reach statistical significance (p>0.05) when the treatment and negative control groups were compared.

Conclusions:

No effects on the induction of MNPCE were observed with the test agent in three different doses for males and females.

Executive summary:

The erythrocyte micronucleus test of the test substance in male and female ICR mice has been studied according to OECD Guideline 474.

25 male and 25 female ICR mice were randomly assigned to 5 groups. The positive controls were treated with cyclophosphamide at the concentration of 40 mg/kg and the negative controls received excipient only. The male and female sample groups were dosed with the test substance at doses of 25, 50 and 100 mg/kg twice, with an interval 24 h. Bone marrow was harvested 20h after the last dosing and analyzed for frequency of MNPCE. A minimum of 2000 polychromatic erythrocytes (PCEs) was analyzed for each animal. The ratio PCE / NCE was determined by counting a minimum of 200 erythrocytes (PCEs plus normachromatic erythrocytes [NCEs] ).

 

No clinical signs were observed in any test animal which could be suspected to be due to the test substance. And no death occurred in any test animal.

A statistically significant increase in the incidence of MNPCE and decrease in the polychromatic erythrocyte fraction over the control value was observed following treatment with the positive control substance cyclophosphamide ( P < 0.01), relative to the negative control in either male or female mouse bone marrow. The values for three treated doses for males and females did not reach statistical significance (p > 0.05) when the treatment and negative control groups were compared.

 

Results indicated that no effects on the induction of MNPCE were observed with the test agent in three different doses for males and females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation:

The mutagenic activity of the test substance was examined in the reverse mutation test by using bacterial strains according to OECD Guideline 471.

The reverse mutation test was composed of the preliminary and the main test, and performed in pre-incubation methods in all bacterial strains in both the presence and the absence of metabolic activation. After the incubation at 37°Cfor 48 hours, number of revertant colonies in each bacterial strain at all dose levels was counted and compared with the negative control.

 

Neither clear dose-related increase in the number of the revertant colonies nor two-fold or more increase in the number of the revertant colonies compared with the negative control was observed in any bacterial strains at any dose levels regardless of the presence or the absence of metabolic activation. And the reappearance of the test results between the preliminary and the main test was confirmed. The values of the negative controls and the positive controls were appropriate in comparison with the historical data of our laboratory. Furthermore, all of the positive controls, such as 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, benzo[α]pyrene and 2-aminoanthracene, increased the number of the revertant colonies two-fold or more compared with the negative control in all bacterial strains, respectively.

 

It is concluded that the mutagenic activity of the test substance is considered negative under the test conditions employed.

Chromosome aberration:

A study was performed to assess the ability of the test substance to induce chromosomal aberrations in human lymphocytes cultured in vitro according to OECD Guideline 473.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition ofphytohaemagglutinin, and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Solvent and positive control cultures were also included. The study comprised two independent tests. In the first test, a three hour treatment was used in both the absence and presence of S9 mix. In the second test, a 21 hour continuous treatment was used in the absence of S9 mix, and a three hour treatment using an increased S9 concentration (5% v/v) was used in the presence of S9 mix. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

 

In both the absence and presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared with the solvent control, in either test.

Silanised glassware was used for the treatment period of all tests. Glassware that had not undergone the silanisation treatment was used to provide a comparison. No significance difference in cytotoxicity or chromosomal aberrations were seen between the two types of glassware.

All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

 

It is included that the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Vitro mammalian cell gene mutation test:

The study was performed to evaluate the test item for its ability to cause gene mutations in vitro in cultured mammalian cells according to OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene).

The mouse lymphoma L5178Y (TK+/--3.7.2C) cells were exposed to six exposure concentrations including 1000, 400, 160, 64, 25.6 and 10.24 μg/mL in the treatment conditions of exposure for 3hours with and without metabolic activation respectively. The cells were treated at six exposure concentrations including 400, 160, 64, 25.6, 10.24 and 4.096 μg/mL in the treatment conditions of exposure for 24 hours without metabolic activation. The positive and solvent (DMSO) controls were included the same time.

 

In this test, the results of the solvent and positive controls met all quality criteria, so the sensitivity of the assay and the efficacy of the S9 mix could be validated.

No test item precipitation was observed at all test concentrations, either at the beginning or the end of treatment.

The results of mutant frequency showed that the induced mutant frequencies (IMF) of all cultures exposed for 3 h and 24 h were consistently less than the average background mutant frequency of 126×10^–6.

 

The results of this study are negative, so it is concluded that the test item is non-mutagenic to the cultured L5178Ymouse lymphoma cells under the conditions of this study.

Mammalian erythrocyte micronucleus test:

The erythrocyte micronucleus test of the test substance in male and female ICR mice has been studied according to OECD Guideline 474.

25 male and 25 female ICR mice were randomly assigned to 5 groups. The positive controls were treated with cyclophosphamide at the concentration of 40 mg/kg and the negative controls received excipient only. The male and female sample groups were dosed with the test substance at doses of 25, 50 and 100 mg/kg twice, with an interval 24 h. Bone marrow was harvested 20h after the last dosing and analyzed for frequency of MNPCE. A minimum of 2000 polychromatic erythrocytes (PCEs) was analyzed for each animal. The ratio PCE / NCE was determined by counting a minimum of 200 erythrocytes (PCEs plus normachromatic erythrocytes [NCEs] ).

 

No clinical signs were observed in any test animal which could be suspected to be due to the test substance. And no death occurred in any test animal.

A statistically significant increase in the incidence of MNPCE and decrease in the polychromatic erythrocyte fraction over the control value was observed following treatment with the positive control substance cyclophosphamide ( P < 0.01), relative to the negative control in either male or female mouse bone marrow. The values for three treated doses for males and females did not reach statistical significance (p > 0.05) when the treatment and negative control groups were compared.

 

Results indicated that no effects on the induction of MNPCE were observed with the test agent in three different doses for males and females.

Justification for classification or non-classification

In accordance with Regulation (EC) No. 1272/2008 section 3.5.2.1 and Table 3.5.1, substances are considered to be classified for germ cell mutagenicity when substances may cause mutations in the germ cells of humans that can be transmitted to the progeny or positive results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo.

As negative results in the bacterial reverse mutation test, chromosome aberration study, vitro mammalian cell gene mutation test and mammalian erythrocyte micronucleus test, therefore this substance should not be classified as a mutagen.