Lithium [ethanedioato-O,O’]tetrafluorophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two in vitro tests are available.

Ames test:

The capability of test item induce reverse mutations in the genome of the histidine requiring tester strains of Salmonella typhimuriumin was evaluated by comparison to negative controls with and without the metabolic activation system (S9 mix), and the method was designed in compliance with OECD 471.Under the conditions of this study, the results of the first test and the validation test were negative. Thus, the test item is considered to be non-mutagenic in the bacterial reverse mutation test using histidine requiring tester strains of Salmonella typhimurium.

In Vitro Mammalian Chromosome Aberration Test:

The ability of test item to induce chromosomal aberrations in human lymphocytes cultured in vitro was assessed based on OECD 473.

The test item has shown evidence of causing an increase in the frequency of structural chromosome aberrations at concentrations of 150 μg/mL, in the in the absence of S9 mix following continuous treatment, and at all test item treatment concentrations (1100, 1300 and 1500 μg/mL) in the presence of S9 mix (5% v/v), in this in vitro cytogenetic test system under the conditions described. In addition, test item has shown significant increases in numerical aberrations in the form of polyploidy, and morphological changes in the form of endoreduplication, in this in vitro cytogenetic test system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2012-05-01 to 2012-09-19

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.: 111202
Purity: 99.5%

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human lymphocytes
- Suitability of cells: Human lymphocytes have been used in this type of study for a number of years.
- Normal cell cycle time (negative control): 14 h

For lymphocytes:
- Sex, age and number of blood donors: two healthy, non-smoking male donors
- Whether whole blood or separated lymphocytes were used: whole blood are used
- Whether blood from different donors were pooled or not: yes
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine

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.
- method of preparation of S9 mix: 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.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL of S9 mix (2% v/v final concentration)

Test concentrations with justification for top dose:

- First test: 20.35, 33.92, 56.52, 94.21, 157.02, 261.70, 436.17, 726.95, 1211.58 and 2019.3 μg/mL with or without S9 mix
- Second test:
In the absence of S9 mix: 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600 and 700 μg/mL.
In the presence of S9 mix: 400, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400 and 1500 μg/mL.

Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The test item was soluble in DMSO at 201.93 mg/mL (1M). On dosing a 201.93 mg/mL solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of 2019.3 μg/mL (10 mM), no precipitate was observed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added to cultures at 1% v/v (50 μL per 5 mL culture).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: approximately 48 hours
- Exposure duration/duration of treatment: First test:3 h; Second test: 21 h without S9 mix, 3h with S9 mix
- Harvest time after the end of treatment (sampling/recovery times): 18 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37°C. After a 10 minute period of incubation at 37°C, the suspensions were centrifuged at
500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at approximately 4°C until slide analysis was completed.
- Microscopic examination: The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures, or cultures where there were no signs of cytotoxicity. From these results the concentration causing a decrease in mitotic index of at least 50% (where possible) of the vehicle control value was the highest concentration selected for metaphase analysis.
Intermediate and low concentrations were also selected.
Where no significant decrease in toxicity was observed (i.e. no significant reduction in mitotic index greater than 50%), the maximum concentration tested was the highest concentration to permit metaphase analysis. Lower concentrations were also selected.
The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective. One hundred metaphase figures were examined from each culture. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen. The vernier readings of all aberrant metaphase figures were recorded.
As a result of the observed statistically increase in polyploidy and endoreduplicated cells, the incidence of polyploid and endoreduplicated cells, out of 500 metaphase cells, was also determined quantitatively for all cultures used for chromosomal analysis under the same treatment condition.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic indices

Statistics:

The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

FIRST TEST
- Toxicity data:
In the absence of S9 mix following 3 hour treatment, test item caused a reduction in the mitotic index to 55% of the vehicle control value at 726.95 μg/mL. The concentrations selected for metaphase analysis were 94.21, 436.17 and 726.95 μg/mL.
In the presence of S9 mix (2% v/v final concentration) following 3 hour treatment, test item caused a reduction in the mitotic index to 74% of the vehicle control value at 1211.58 g/mL. The concentrations selected for metaphase analysis were 157.02, 436.17 and 1211.58 μg/mL.
- Genotoxicity results:
In the absence of S9 mix (3 hour treatment):
test item caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any concentration, when compared to the vehicle control.
Statistically significant increases in the proportion of polyploid cells were observed during metaphase analysis at concentrations of 436.17 μg/mL (p<0.01) and 726.95 μg/mL (p<0.001), when compared to the vehicle control. No statistically significant increases were observed at the lowest analysed concentration of 94.21 μg/mL, when compared to the vehicle control. As a result of the observed statistically significant increases in polyploidy, the incidence of polyploid metaphase cells, out of 500 metaphase cells (where possible), was also determined quantitatively for all cultures in used in chromosomal analysis under the same treatment condition.
The positive control (Mitomycin C) caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system.
In the presence of S9 mix (3 hour treatment):
test item caused a statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at 1211.58 μg/mL (including gaps only), when compared to the vehicle control. However, as the observed increase was within the laboratory historical control range, when taken at the 99% confidence limit, this increase was considered to have been exaggerated by the low vehicle control values, and therefore considered biologically non-relevant. No statistically significant increases were observed at the lower analysed concentrations of 157.02 μg/mL or 436.17 μg/mL, when compared to the vehicle control.
A statistically significant increase in the proportion of polyploidy cells were observed during metaphase analysis at 1211.58 μg/mL (p<0.001), when compared to the vehicle control. No statistically significant increases were observed at the lower analysed concentrations of 157.02 μg/mL and 436.17 μg/mL, when compared to the vehicle control.
A statistically significant increase in the proportion of endoreduplicated cells was observed during metaphase analysis at 1211.58 μg/mL only (p<0.001), when compared to the vehicle control. No statistically significant increases were observed at the lower tested concentrations of 157.02 μg/mL and 436.17 μg/mL, when compared to the vehicle control.
As a result of the observed statistical increases in polyploidy and endoreduplication, the incidence of polyploid and endoreduplicated cells, out of 500 metaphase cells (where possible), was also determined quantitatively for all cultures in used in chromosomal analysis under the same treatment condition.
The positive control (Cyclophosphamide) caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

SECOND TEST
- Toxicity data:
In the absence of S9 mix following 21 hour continuous treatment, test item caused a reduction in the mitotic index to 48% of the vehicle control value at 150 μg/mL. The concentrations selected for metaphase analysis were 25, 75 and 150 μg/mL.
In the presence of S9 mix (5% v/v final concentration) following 3 hour treatment, test item caused a reduction in the mitotic index to 54% of the vehicle control value at 1500 μg/mL. The concentrations selected for metaphase analysis were 1100, 1300 and 1500 μg/mL.
- Genotoxicity results:
In the absence of S9 mix (21 hour continuous treatment):
test item caused statistically significant increases (p<0.001: including and excluding gaps) in the proportion of cells with chromosomal aberrations at 150 μg/mL only, when compared to the vehicle control. No statistically significant increases were observed at the lower analysed concentrations of 25 μg/mL or 75 μg/mL, when compared to the vehicle control. All mean values for the vehicle control (DMSO) and the 25 μg/mL and 75 μg/mL test item treatment concentrations were within the laboratory historical control range, when taken at the 99% confidence limit. However, at 150 μg/mL, the highest analysed concentration, statistically significant increases (p<0.001) were observed in the proportion of aberrant cells, which clearly exceeded the laboratory historical control range. No statistically significant increases in the proportion of polyploid or endoreduplicated cells were observed during metaphase analysis at any concentration, when compared to the vehicle control.
The positive control (Mitomycin C) caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system.
In the presence of S9 mix (3 hour treatment):
test item caused statistically significant increases (p<0.001: including and excluding gaps) in the proportion of cells with chromosomal aberrations at all analysed concentrations (1100, 1300 and 1500 μg/mL), when compared to the vehicle control. All mean values for the vehicle control (DMSO) were within the laboratory historical control range, when taken at the 99% confidence limit. All test item treatment concentration analysed clearly exceeded the laboratory historical control range.
Statistically significant increases in the proportion of polyploidy cells were observed during metaphase analysis at 1100 (p<0.001), 1300 μg/mL (p<0.001) and 1500 μg/mL (p<0.01), when compared to the vehicle control. A statistically significant increase in the proportion of endoreduplicated cells was observed during metaphase analysis at 1100 μg/mL (p<0.001) and 1300 μg/mL (p<0.01), when compared to the vehicle control.
As a result of the observed statistical increase in the incidence of polyploidy and endoreduplicated cells during metaphase analysis, the incidence of polyploid and endoreduplicated cells, out of 500 metaphase cells (where possible), was also determined quantitatively for all cultures in used in chromosomal analysis under the same treatment condition.
The positive control (Cyclophosphamide) caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Conclusions:

It is concluded that the test item has shown evidence of causing an increase in the frequency of structural chromosome aberrations at concentrations of 150 μg/mL, in the in the absence of S9 mix following continuous treatment, and at all test item treatment concentrations (1100, 1300 and 1500 μg/mL) in the presence of S9 mix (5% v/v), in this in vitro cytogenetic test system under the conditions described. In addition, the test item has shown significant increases in numerical aberrations in the form of polyploidy, and morphological changes in the form of endoreduplication, in this in vitro cytogenetic test system.

Executive summary:

The ability of test item to induce chromosomal aberrations in human lymphocytes cultured in vitro was assessed based on OECD 473.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin, and exposed to LiPF4(Ox) both in the absence and presence of S9 mix derived from rat livers. Vehicle and positive control cultures were also included.

The study comprised of two independent tests. In the first test, a 3 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 3 hour treatment using an increased S9 concentration (5% v/v) was used in the presence of S9 mix

The test concentrations are listed as follows:

First test:20.35, 33.92, 56.52, 94.21, 157.02, 261.70, 436.17, 726.95, 1211.58 and 2019.3 μg/mL with or without S9 mix

Second test: Without S9 mix: 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600 and 700 μg/mL; with S9 mix: 400, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400 and 1500 μg/mL.

The test item has shown evidence of causing an increase in the frequency of structural chromosome aberrations at concentrations of 150 μg/mL, in the in the absence of S9 mix following continuous treatment, and at all test item treatment concentrations (1100, 1300 and 1500 μg/mL) in the presence of S9 mix (5% v/v), in this in vitro cytogenetic test system under the conditions described. In addition, test item has shown significant increases in numerical aberrations in the form of polyploidy, and morphological changes in the form of endoreduplication, in this in vitro cytogenetic test system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-01-10 to 2020-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: 190805
Purity: >99%

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

TA97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : prepared in-house
- method of preparation of S9 mix: 10% S9 mix was prepared immediately before the test and placed in a mixture of water and ice during the test.
- concentration or volume of S9 mix and S9 in the final culture medium: 500 μL
- quality controls of S9: Before being used, this batch of S9 was tested for its sterility, its protein content (not higher than 40 mg/ml), and its capability to activate known mutagens in Ames test. The results of the quality test indicated that each index of S9 was in the expected range to meet the test requirements.

Test concentrations with justification for top dose:

FIRST TEST and VALIDATION TEST: 5000, 1500, 500, 150, 50 and 15μg/plate without S9 mix and with S9 mix

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: chosed based on solubility results of the test item

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: Dexon: For TA97a, TA98 and TA102 without S9 mix (S9-); 2-Aminofluorene (2-AF): For TA97a, TA98 and TA100 with S9 mix (S9+); 1, 8-Dihydroxyanthraquinone (Danthron): For TA102 in S9+; 2-Aminoanthracene (2-AA): For TA1535 in S9+

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.9~9×109 CFU/mL
- Test substance added in medium; in agar (plate incorporation) for the first test; preincubation for the validation test

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: validation test: a minimum of 20min
- Exposure duration/duration of treatment: first test: about 66 hours; validation test: about 67 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: numbers of revertant colonies and signs of background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY
After incubation, the number of revertant colonies in each plate was counted. As counting the plates of the positive controls (except for TA1535), a plate was divided into eight sectors on the back, and two diagonal sectors were chosen randomly and counted, then the result was multiplied by 4 as the number of revertant colonies

Evaluation criteria:

- CRITERIA OF CYTOTOXICITY
The test item is evaluated as cytotoxicity if one of the below criteria is met.
1) Compared with the concurrent solvent control, the number of the revertant colonies has significant decrease or is none;
2) Compared with the concurrent solvent control, the signs of the background lawn are thin or clearing.
- CRITERIA OF POSITIVE RESULT
1) When there is a concentration-related increase over the range (equal to or greater than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and equal to or greater than three times of that of the concurrent solvent control in TA1535) in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.
2) When there is a reproducible increase (equal to or greater than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and equal to or greater than three times of that of the concurrent solvent control in TA1535) at one or more concentrations in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.
- CRITERIA OF NEGATIVE RESULT
The test item should be evaluated as negative if none of the above criteria is met.
- CRITERIA OF EQUIVOCAL RESULT
Although most tests give clear positive or negative results, in some instances the data generated prohibit test item from making a definite judgment. Results of this type was reported as equivocal.

Species / strain:

other: TA97a, TA98, TA100, TA102 and TA1535

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- Test conditions: The standard plate incorporation method was performed at 3 dose levels, including 5000, 1000 and 200μg/plate, in five tester strains of TA97a, TA98, TA100, TA102 and TA1535, with and without metabolic activation system. Solvent control (DMSO) in each tester strain was performed at the same time. The dose volume of each dose group and solvent control group were 0.1ml/plate, in duplicate.
- Preliminary test results: About the precipitate of the test item, monitoring of TA98 showed that there was no precipitate at any designed dose level before and after incubation with and without metabolic activation system. Moreover, compared to the concurrent solvent controls, the test item was considered to be not cytotoxic to any tester strains at any designed dose levels with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
In the first test and the validation test, the mean number of revertant colonies in the solvent controls and positive controls were within the range of historical control data in this lab. In addition, the background lawn in the solvent controls and positive controls had grown as densely packed microcolonies which form a uniform layer observed with microscope.

Ames test:
- Signs of toxicity : In the first test, no cytotoxicity was observed at any designed dose level in all tester strains in two treatment conditions. It was showed that Tthe numbers of revertant colonies has not significant decrease, and the background lawn was not thinner than the concurrent solvent controls. In the validation test, the same cytotoxicity results were observed as in the first test.
- Individual plate counts : The results of the viable count in two tests showed that the density of the cultures for each tester strain were within the acceptable range of 0.9~9×109 CFU/mL.
- Mean number of revertant colonies per plate and standard deviation : In the first test, the mean number of revertant colonies at each dose level was less than two times of that of the concurrent solvent controls in TA97a, TA98, TA100, TA102 and less than three times of that of the concurrent solvent controls in TA1535. In the validation test, the same mutagenic result was obtained as in the first test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Refer to The database of the historical controls data for the tester strains (2019) in attached background material.

Conclusions:

Under the conditions of this study, the results both in the first test and the validation test were negative. Thus, the test item considered to be non-mutagenic in the bacterial reverse mutation assay using the histidine requiring tester strains of Salmonella typhimurium.

Executive summary:

The capability of test item induce reverse mutations in the genome of the histidine requiring tester strains of Salmonella typhimuriumin was evaluated by comparison to negative controls with and without the metabolic activation system (S9 mix), and the method was designed in compliance with OECD 471.

Five histidine requiring (his-) mutant tester strains of Salmonella typhimurium including TA97a, TA98, TA100, TA102 and TA1535 were treated with test item using the standard plate incorporation method and the pre-incubation method at six dose levels (5000, 1500, 500, 150, 50 and 15 µg/plate), in triplicate, with the solvent controls and positive controls, both in the presence and absence of the S9 mix.

 

In the first test, there was no precipitate on the surface of MGA plate at any dose level before and after the TA98 incubation, with and without metabolic activation system. In addition, no cytotoxicity was observed at any designed dose level in all tester strains in two treated conditions. In the validation test, the same results of the precipitate and cytotoxicity were observed as that in the first test.

In the first test, with and without metabolic activation, the mean number of revertant colonies at each dose level was less than two times of that of the concurrent solvent control in TA97a, TA98, TA100, TA102 and less than three times of that of the concurrent solvent control in TA1535. In the validation test, the same mutagenic results were obtained as that in the first test.

Under the conditions of this study, the results of the first test and the validation test were negative. Thus, the test item is considered to be non-mutagenic in the bacterial reverse mutation test using histidine requiring tester strains of Salmonella typhimurium.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus test:

The potential induction of micronuclei by test item in bone marrow cells of CD1 mice was assesses according to OECD 474.The test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female CD1 mice when administered orally by gavage in this in vivo test procedure.

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 2013-01-09 to 2013-04-10

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:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Batch No.: 111202
Purity: 99.5%

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Young adult animals are chosen for use because of the high rate of cell division in the bone marrow, the wealth of background data on this species, and their general suitability for toxicological investigations.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: ca 40-47 days old.
- Weight at study initiation: Males weighed between 27.4 g to 32.1 g. Females weighed between 21.7 g to 25.5 g.
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Each group was kept with the sexes separated in cages and maintained in a controlled environment
- Diet: pelleted expanded rat and mouse No.1 maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 5, 10, 20, 30 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg.
- Lot/batch no.: MKBF8603V

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test substance were prepared corn oil.
Positive control solution was prepared using purified water at a concentration of 0.6 mg/mL just prior to administration

Duration of treatment / exposure:

administered on two occasions approximately 24 hours apart

Frequency of treatment:

twice

Dose / conc.:

12.5 mg/kg bw/day (actual dose received)

Remarks:

For males

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

For males and females

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

For males and females

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

For females

No. of animals per sex per dose:

5 male/female for loe and mid dose, 7 male or 7 female for high dose group

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Route of administration: oral
- Doses / concentrations: dosed once at 12 mg/kg/day

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
All the animals were killed by exposure to rising levels of carbon dioxide and both femurs dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.

DETAILS OF SLIDE PREPARATION:
The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides

METHOD OF ANALYSIS:
Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.

Evaluation criteria:

incidence of micronucleated polychromatic erythrocytes for the treatment group compared with the vehicle control group (p<0.05); individual and/or group mean values should exceed the laboratory historical control range.
A negative result is indicated where individual and group mean incidences of micronucleated polychromatic erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent vehicle control group and where these values fall within the historical control range.
An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant decrease in the proportion of polychromatic erythrocytes (p<0.05).

Statistics:

For the proportion of polychromatic erythrocytes at 24 hours in male animals, an asymptotic one-tailed Jonckheere’s test for trend with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests, for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 6.
For the proportion of polychromatic erythrocytes at 24 hours in female animals, an asymptotic one-tailed Jonckheere’s test for trend with “step-down” was used on Groups 1 to 5 (excluding group 2) for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 4 (excluding group 2), then on Groups 1 and 3 (excluding group 2). Exact one-tailed Wilcoxon pairwise tests, for a decrease from control, were also carried out on Group 1 (control) versus Groups 3, 4, 5 and 6.
For incidences of micronucleated polychromatic erythrocytes at 24 hours in males, an exact one-tailed Linear-by-Linear association test with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Also, exact one-tailed pairwise Permutation tests, for an increase from control, were carried out on Group 1 (control) versus Groups 2, 3, 4 and 6.
For incidences of micronucleated polychromatic erythrocytes at 24 hours in females, an exact one-tailed Linear-by-Linear association test with “step-down” was used on Groups 1 to 5 (excluding group 2) for an increase from control. If significant, then the analysis was carried out on Groups 1 to 4 (excluding group 2). Also, exact one-tailed pairwise Permutation tests, for an increase from control, were carried out on Group 1 (control) versus Groups 3, 4, 5 and 6.
Statistical significance was declared at the 5% level for all tests.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 50-300 mg/kg/day
- Clinical signs of toxicity in test animals:
To determine suitable dose levels for use in the micronucleus test, a group consisting of two male and two female animals were administered test item at 100 mg/kg/day on two consecutive days approximately 24 hours apart.
At 100 mg/kg/day, clinical signs of toxicity were observed in one male animal only on Day 3. Clinical signs of toxicity included hindlimbs splayed, underactive behaviour, hunched posture, slow breathing and both eyelids partially closed.
On the basis of this result it was considered that the maximum tolerated dose (MTD) had not been achieved, therefore, an additional group of two male and two female animals were administered test item at 300 mg/kg/day.
At 300 mg/kg/day, clinical signs of toxicity were observed on Day 1, these included piloerection, underactive behaviour, hunched posture, slow breathing, hindlimbs splayed, reduced body temperature, salivation, prostrate posture, gasping breathing and moderate tremors. Due to the severity of the clinical signs observed the animals were killed in extremis.
On the basis of this result the MTD had been exceeded, therefore an additional group of two male and two female animals were administered test item at 200 mg/kg/day.
At 200 mg/kg/day, clinical signs of toxicity observed included underactive behaviour, hindlimbs splayed, hunched and prostrate posture, both eyelids partially closed, unsteady gait, uncoordinated gait, vocalisation, slow and irregular breathing and moderate tremors. One male animal was found dead on Day 1, the remaining male and two female animals were killed in extremis due to the severity of the clinical signs observed.
On the basis of this result the MTD had been exceeded. As a result of the inconsistent findings observed between animals previously administered test item at 100 mg/kg/day an additional group of two male and two female animals were administered test item at 100 mg/kg/day.
At 100 mg/kg/day, clinical signs of toxicity observed in male animals on Day 1 included underactive behaviour, piloerection, hunched posture, hindlimbs splayed, reduced body tone, and temperature, shallow breathing, slow breathing and urine staining. Due to the severity of the clinical signs observed both animals were killed in extremis.
At 100 mg/kg/day, clinical signs of toxicity observed in female animals on Day 1 included underactive behaviour, piloerection, both eyelids partially closed and hunched posture. The animals survived until scheduled termination with no clinical signs of toxicity displayed on Day 2 or 3.
On the basis of this result, the MTD had been exceeded for male animals. Therefore an additional group of two male animals were administered test item at 50 mg/kg/day. At 50 mg/kg/day, underactive behaviour was observed on Day 3 only.

- High dose with and without activation: the MTD in male animals was considered to be 50 mg/kg/day and the MTD in female animals was considered to be 100 mg/kg/day.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
Micronucleated polychromatic erythrocyte counts (MPCE): The test item did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes in male or female CD1 mice.
Micronucleated normochromatic erythrocytes (MNCE): The test item did not cause any significant increases in the incidence of micronucleated normochromatic erythrocytes in male or female CD1 mice.
- Proportion of polychromatic erythrocytes (%PCE): did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male or female CD1 mice.
- Appropriateness of dose levels and route:
The oral route was chosen for this particular study as to maximise exposure to the test system.
Animals were treated with test item at dose levels of 12.5, 25 and 50 mg/kg/day for male animals and 25, 50 and 100 mg/kg/day for female animals.No mortalities were observed during the micronucleus test. No clinical signs of toxicity were observed for the vehicle control and positive control or male animals administered test item at 12.5 mg/kg/day and female animals administrated test item at 25 and 50 mg/kg/day over the duration of the test.
At 25 mg/kg/day, one male animal displayed piloerection on Day 2 only. At 50 mg/kg/day, piloerection was observed in male animals. At 100 mg/kg/day, piloerection was observed in female animals. Some incidences of bodyweight loss were observed during the micronucleus test

-Positive Control:

Mitomycin C caused a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (p<0.01) in male and female CD1 mice.

Mitomycin C did not cause a statistically significant decrease in the proportion of polychromatic erythrocytes in male or female CD1 mice.

Conclusions:

The test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female CD1 mice when administered orally by gavage in this in vivo test procedure.

Executive summary:

The potential induction of micronuclei by test item in bone marrow cells of CD1 mice was assesses according to OECD 474. Animals were treated with test item orally by gavage on two occasions approximately 24 hours apart. A substantial difference in toxicity was observed between the sexes in the preliminary toxicity test. In line with current guidelines the micronucleus test was performed using male and female animals. In the micronucleus test male animals were administered test item at 12.5, 25 and 50 mg/kg/day, female animals were administered test item at 25, 50 and 100 mg/kg/day.

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes were observed in male or female CD1 mice treated with test item at any treatment level, compared to vehicle control values. 

It is concluded that the test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female CD1 mice when administered orally by gavage in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Data:

In vitro:

Ames: OECD 471, negative

In Vitro Mammalian Chromosome Aberration Test: OECD 473, positive

In vivo:

In vivo micronucleus test: OECD 474, negative

 

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the test substance should not be classified as germ cell mutagens as negative result arrived in vivo micronucleus test.