Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the key bacterial reverse mutatioin assay according to OECD guideline 471, the results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.


 


In the key in vitro mammalian chromosome aberration test according to OECD guideline 473, the test material was considered to be non-clastogenic to human lymphocytes with and without metabolic activation.


 


In the key in vitro mammalian cell gene mutation test according to OECD guideline 476, the test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was not considered to be mutagenic under the conditions of the test.


 


Conclusion: The test item was neither clastogenic, not mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-10 to 2010-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
No. 440/2008 of 30 May 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase heterozygous system, TK +/- to TK -/- conversion
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK +/- 3.7.2c mouse lymphoma cell line, obtained from Dr. J. Cole of the MRC Cell Mutation Unit, University of Sussex, Brighton, UK
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
0, 6.09, 12.19, 24.38, 48.75, 97.5, 195, 390, 780, 1560 µg/mL

Main Experiment, 4-Hour, Without S9:
0, 12.5, 25, 50, 100, 125, 150, 175, 200 µg/mL

Main Experiment, 4-Hour, With S9:
0, 25, 50, 100, 125, 150, 175, 200, 250 µg/mL

Main Experiment, 24-Hours, Without S9:
0, 6.25, 12.5, 25, 50, 75, 100, 125, 150 µg/mL
Vehicle / solvent:
Dimethyl sulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9 activation: Sigma batch 1419706 and 15108051 at 400 µg/mL (4 hour) and 150 µg/mL (24-hour)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 activation: Acros batch A0164185 at 2 µg/mL
Details on test system and experimental conditions:
Cell Culture

Cells were stored in liquid nitrogen at -196°C. Cells were cultured at 37°C with 5% CO2 in air in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media). Cells had a generation time of approximately 12 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) were used during the course of the study. Master cultures of stock cells were checked and found to be mycoplasma free.

Preparation of test and control materials

The test material was dissolved in dimethyl sulfoxide (DMSO) and the appropriate dilutions were made. The maximum dose level investigated in the preliminary toxicity test was 1560 μg/mL which was equivalent to approximately 10 mM. Analysis for concentration, homogeneity and stability of the test material preparations were not a requirement of the test method and were therefore not performed.

Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) at 400 μg/mL and 150 μg/mL for the 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) 2 μg/ml was used as the positive control in the presence of metabolic activation.

Preliminary toxicity test

A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 6.09 to 1560 μg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 2 x 10^5 cells/mL.

The cultures were incubated and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL. After a further 24 hours, the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth values (SG). The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth Value (%RSG).

Results from the preliminary toxicity test were used to set the test material dose levels for the mutagenicity experiment. Maximum dose levels were selected using the following criteria:

i) Maximum recommended dose level, 5000 μg/ml or 10 mM.
ii) The presence of excessive precipitate where no test material-induced toxicity was observed.
iii) Test material-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).

Mutagenicity test (main test)

4-Hour exposures, with and without metabolic activation

An exponentially growing stock culture of cells was set up to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/ml in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test material (12.5 to 200 μg/mL in the absence of metabolic activation, and 25 to 250 μg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.

The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

24-Hour exposures, without metabolic activation

Cells were counted and processed (as above) to give 0.3 x 10^6 cells/mL in 10 mL duplicate cultures established in 25 cm2 tissue culture flasks. To each culture was added 2 mL of the treatment dilutions (0.2 mL for the positive control) and sufficient R10 medium to give a final volume of 20 mL. The dose range of the test material was 6.25 to 150 μg/mL. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker for 24 hours.

Cell processing following treatment periods

At the end of the treatment periods for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL.

On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate scoring

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies were scored manually by eye using qualitative judgment. Large colonies were defined as those covering approximately ¼ to ¾ of the surface of the well and were generally no more than one or two cells thick. As a rule of thumb, all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies were normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. the MTT vital stain was taken up by viable cells to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-200 x 10^-6 for the TK+/- locus in L5178Y cells at this testing laboratory. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10^-6 mutant frequency per survivor are not normally acceptable and will be repeated. Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. A Global Evaluation Factor (GEF) value was set following International Workshop (Moore et al., 2003; Moore et al, 2006) at 126 x 10^-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgment will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Robinson W D et al (1989) Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp102-140.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The maximum dose level used was limited by test material toxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test

The results for Relative Suspension Growth (%RSG) are as shown in Table 1:

A precipitate of the test material was observed at and above 195 μg/mL in the 4-hour exposure group in the absence of metabolic activation, and at and above 390 μg/ml in both the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation. The precipitate was observed to become greasy and oily in appearance at 1560 μg/mL in both of the 4-hour exposure groups.

Mutagenicity test results

4-Hour exposures with and without metabolic activation (Table 2):

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any significant reductions in (%V) viability in either the absence or presence of metabolic activation, therefore indicating that no residual toxicity had occurred. Near optimum levels of toxicity were achieved in the absence of metabolic activation. Optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation due to the sharp onset of toxicity, despite using a very narrow dose interval. However, a dose level that exceeded the usual acceptable upper limit of toxicity was plated for viability and TFT resistance for each of the exposure groups. It was therefore considered that with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including the dose levels that exceeded the usual upper limit of acceptable toxicity, or in the 24-hour exposure group where optimum levels of toxicity were achieved, the test material had been adequately tested. The excessive toxicity observed at 200 μg/mL in the absence of metabolic activation, and at 250 μg/mL in the presence of metabolic activation, resulted in these doses not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.

24-Hour exposure without metabolic activation (Table 3):

As was seen in the Preliminary Toxicity Test, there was evidence of a marked reduction in % RSG and RTG values in cultures dosed with the test material. There was also evidence of a modest reduction in (%V) viability, therefore indicating that residual toxicity had occurred. Optimum levels of test material-induced toxicity were achieved. The positive control induced acceptable levels of toxicity.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had an effect on the toxicity of the test material. The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

The test material induced a small statistically significant dose related (linear-trend) in the mutant frequency x 10^-6 per viable cell (Table 9). However, statistically significant increases in mutant frequency were not observed at any of the individual dose levels, the GEF was not exceeded at any of the individual dose levels, and the mutant frequency values observed were within the acceptable range for vehicle controls. Therefore, the response was considered to be spurious and of no toxicological significance. Precipitate of the test material was not observed at any of the dose levels.

Table 1: Preliminary Toxicity Test Results


 




















































































Dose


(mg/mL)



% RSG (-S9)


4-Hour Exposure



%RSG (+ S9)


4-Hour Exposure



%RSG (-S9)


24-Hour Exposure



 



 



 



 



0



100



100



100



6.09



93



103



94



12.19



87



116



81



24.38



87



111



80



48.75



90



103



62



97.5



84



101



34



195



0



28



0



390



0



0



0



780



0



0



0



1560



0



0



0



 



 



 



 



 


 


Table 2: Experimental Results


 
























































































































































































4-Hour Test Without S9



 



 



 



 



 



 



Treatment


(mg/mL)



%RSG



RTG



MF



 



 



 



 



0



100



1.00



118.34



12.5*



95



 



 



25



92



1.16



91.83



50



88



1.05



101.08



100



84



0.99



104.88



125



73



0.81



120.90



150



37



0.38



122.92



175**



1



0.03



143.07



200***



0



-



-



Linear trend



 



 



NS



EMS



 



 



 



400



74



0.60



684.79



4-Hour Test With S9



 



 



 



 



 



 



0



100



1.00



149.96



12.5*



92



 



 



25



90



0.98



91.37



50



83



0.88



147.57



100



83



0.97



150.12



125



77



0.81



106.33



150



50



0.50



116.61



175**



9



0.12



111.16



200*



0



-



-



Linear trend



 



 



NS



CP



 



 



 



2



48



0.24



781.04



* Not plated for viability or 5-TFT resistence.


** Treatment excluded from statistics due to toxicity.


 


Table 3: Experimental Results


 





































































































24-Hour Test Without S9



 



 



 



 



 



 



Treatment


(mg/mL)



%RSG



RTG



MF



 



 



 



 



0



100



1.00



115.95



6.25*



104



 



 



12.5



108



0.92



91.35



25



99



0.96



98.55



50



71



0.66



121.85



75



46



0.43



101.71



100



30



0.16



149.79



125



11



0.10



158.82



150*



2



-



-



Linear trend



 



 



p < 0.05



EMS



 



 



 



150



66



0.45



1007.15



* Not plated for viability or 5-TFT resistence.


 

Conclusions:
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was not considered to be mutagenic under the conditions of the test.
Executive summary:

The GLP compliant study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls. The exposure groups used were as follows: 4-hour exposures both with and without metabolic activation, and 24 hours without metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test and was 12.5 to 200 |jg/ml for the 4-hour exposure group in the absence of metabolic activation, 25 to 250 |jg/ml for the 4-hour exposure group in the presence of metabolic activation, and 6.25 to 150 \ig/m\ for the 24-hour exposure group in the absence of metabolic activation. The maximum dose level used was limited by test material-induced toxicity. Precipitate of test material was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-07-19 to 2000-08-25
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
Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100, TA102) and frameshift (TA1537, TA98) mutations.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Source of S9: King & Harnasch GmbH (lot KH3799), 79199 Kirchzarten
- Method of preparation of S9 mix: S9 was prepared according to the method recommended by Ames et al. (1975). Sprague-Dawley male rats (8-10 weeks old) induced with Aroclor 1254 (500 mg/kg body weight) were used to prepare the 9000g supernatant of liver homogenates (S9). S9-mix containing 10% S9 was freshly prepared for each mutagenicity assay. The concentration of the cofactors in the S9-mix (per mL) were: 0.335 mL distilled water, 0.5 mL 0.2 M and pH 7.4 phosphate buffer, 0.04 mL 0.1 M NADP, 0.005 mL 1 M glucose-6-phosphate, 0.02 mL MgCl2/KCl (0.4M/1.65M), 0.1 mL S9 fraction.
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (containing 0.05 mL S9) in the final culture medium (2.7 mL in total)
- Quality controls of S9: Enzymatic activity (using benzo(a)pyrene and 2-aminoanthracene), sterility
Test concentrations with justification for top dose:
Concentrations between 5 and 5000 µg/plate were used in both experiment I and II in the presence and absence of S9. The top dose was chosen according to OECD guideline 471 and according to an initial toxicity test.
Vehicle / solvent:
- Vehicles used: DMSO (test item, 2-aminoanthracene, 2-nitrofluorene), distilled water (9-aminoacridine, mitomycin c, sodium azide)

- Justification for choice of solvent/vehicle: Solubility properties
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (test item, 2-aminoanthracene, 2-nitrofluorene), distilled water (9-aminoacridine, mitomycin c, sodium azide)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Remarks:
9-AA
2-NF
SAZ
MIT C
2-AA
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: 1-3x10^9 cells/mL
- Test substance added in: Agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE
- Exposure duration/duration of treatment: 48-72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colony numbers
Rationale for test conditions:
Based on an initial toxicity test
Evaluation criteria:
A result is considered positive if the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the treatment at any dosage level reveals a significant effect at any of the test points.
Statistics:
Estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a X2-test.
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
True negative controls validity:
not examined
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
True negative controls validity:
not examined
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
True negative controls validity:
not examined
Positive controls validity:
valid
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
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test compound on the plates was not observed.
- Definition of acceptable cells for analysis: When there was any question about the nature of colonies scored as revertants and when positive mutagenic results are obtained, the genotype of revertant colonies are spot-checked by picking and streaking on histidine free plates.

RANGE-FINDING/SCREENING STUDIES
A initial toxicity test was conducted to determine concentrations for the main test. Concentration between 5 and 5000 µg/plate were consequently chosen for the main test.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Ames test:
- Signs of toxicity: In the absence of S9-mix, the test item was bacteriotoxic towards the strains TA98, TA102, and TA1537 at 1500 µg/plate and towards the strains TA100 and TA1535 at 5000 μg/plate. In the presence of S9-mix, the test item was bacteriotoxic towards all strains at 500 µg/plate.
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative historical control data: See "Attached background material"
Conclusions:
In a bacterial reverse mutation assay according to OECD guideline 471, the results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.
Executive summary:

In a GLP compliant study according to OECD guideline 471, the mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test item was dissolved in DMSO and tested in concentrations of 5 to 5000 µg per plate in the presence and absence of S9. In the absence of S9-mix, the test item was bacteriotoxic towards the strains TA98, TA102, and TA1537 at 1504g/plate and towards the strains TA100 and TA1535 at 5000n/plate. In the presence of S9-mix, the test item was bacteriotoxic towards all strains at 5000 µg/plate. Precipitation of the test compound on the plates was not observed. Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system. In the concentration range investigated, the test item did not induce any increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-14 to 2010-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Primary human lymphocytes received from volunteer who had been previously screened for suitability.
- Normal cell cycle time: The cell-cycle time was determined using BrdU incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.

For lymphocytes:
- Number of blood donors: One single donor
- Whether whole blood or separated lymphocytes were used: Whole blood culture was used
- Whether blood from different donors were pooled or not: Blood from a single donor was used.
- Mitogen used for lymphocytes: Demecolcine (Colcemid 0.1 µg/mL)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal calf serum, at 37°C with 5% C02 in air.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Mitosis was arrested by addition of demecolcicine (0.1 µg/mL) two hours before the required harvest time.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Source of S9: Livers of male Sprague-Dawley rats weighing approximately 250 g. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg bw) and p-naphthoflavone (100 mg/kg bw).
- Method of preparation of S9 mix: Not indicated
- Concentration or volume of S9 mix and S9 in the final culture medium: 1 mL of 20% of S9 mix (containing 2% S9) in the final culture medium (10.1 mL)
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 6.10, 12.19, 24.38, 48.75, 97.5, 195, 390, 780 and 1560 µg/mL;

Main Experiment, 4-hour treatment period with or without S9, followed by a 20-hour treatment free period: 0, 100, 200, 300, 400, 600 and 800 µg/mL;

Main Experiment, without S9, 24-hour treatment period: 0, 12.5, 25, 50, 100, 200 and 300 µg/mL


The test concentrations were chosen based on a preliminary toxicity test where concentrations of up to 1560 µg/mL (equivalent to 10 mM) were tested (10 mM= maximum recommended dose level according to OECD guideline 473).
Vehicle / solvent:
- Vehicles used: DMSO (test item, cyclophosphamide), cell culture medium (mitomycin c)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (CP), cell culture medium (MMC)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Without S9: MMC (0.4 and 0.2 µg/mL);
With S9: CP (5 µg/mL)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 3 parallel cultures with three different treatment conditions

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in: Medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4-hour exposure/20-hour expression period with S9 (experiment I), 4-hour exposure/20-hour expression period without S9 (experiment II), 24-hour continuous exposure without S9 (experiment III)
- Harvest time after the end of treatment: Harvest after a 20-hour of recovery period after 4 hours of treatment (experiment I+II), harvest after 24 hours of treatment without recovery period (experiment III)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Demecolcine (Colcemid 0.1 µg/mL) colchicine) was added two hours before the required harvest time.
- Methods of slide preparation and staining technique used including the stain used: The lymphocytes were re-suspended in fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. When the slides were dry, they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded. Where possible, the first 100 consecutive well-spread metaphases from each culture were counted. Where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells.
- Criteria for scoring micronucleated cells: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.
- Methods to characterize whether micronuclei contain whole or fragmented chromosomes: If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): See "Chromosome Structural Aberrations: Classification, Evaluation Criteria" under "Any other information on material and methods, incl. tables"
- Determination of polyploidy: Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Mitotic index (MI)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value.
Rationale for test conditions:
Test conditions were chosen on the basis of the methods described in OECD guideline 473.
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There was no significant change in pH when the test material was dosed into media.
- Data on osmolality: The osmolality did not increase by more than 50 mOsm.
- Possibility of evaporation from medium: The test material was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration.
- Precipitation and time of the determination: Precipitation was not seen in any of the exposure groups at the end of exposure.

RANGE-FINDING/SCREENING STUDIES
A preliminary experiment was conducted to check for toxicity and precipitation. A greasy/oily precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 390 µg/mL, in experiment I and III. In experiment II, a greasy/oily precipitate was seen at and above 780 µg/mL. Hemolysis was seen at and above 97.5 µg/mL at the end of exposure in the 4/20-hour and 24-hour exposure groups in the absence of S9 and at and above 48.75 µg/mL in the 4/20-hour exposure group with S9. Microscopic analysis of slides prepared from the exposed cultures indicated metaphase cells were present up to 390 µg/mL in the 4/20-hour exposures with and without S9. In the 24-hour cultures, the maximum dose at which metaphases were present was 195 µg/mL. The selection of the maximum dose level for the main experiment was based on toxicity and was 800 µg/mL for both experiment I+II and 300 µg/mL for experiment III.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Control culture aberration frequencies were acceptable and were within historical control ranges for in-house data, see "Attached background material"

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
o For lymphocytes in primary cultures: mitotic index (MI): See "Attached background material"
- Genotoxicity results
o Definition for chromosome aberrations, including gaps: See "Any other information on material and methods, incl. tables"
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps: See "Attached background material"

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle) historical control data: See "Attached background material"
- Positive historical control data: See "Attached background material"
Conclusions:
The test material did not induce a statistically significant increase in the frequency of cells with chromosomal aberrations either with or without rat liver S9 metabolic activation. The test material was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

This GLP compliant report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24 hours continuous exposure in the absence of metabolic activation. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included a dose level that induced near optimum mitotic inhibition in the 4(20)-hour exposure group in the presence of S9 and in the 24 hour exposure group. Optimum toxicity was not achieved in the 4(20)-hour exposure group in the absence of S9 due to the steepness of the toxicity curve. The test material was considered to be non-clastogenic to human lymphocytes in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay, RL1


In a GLP compliant study according to OECD guideline 471, the mutagenicity of the test substance was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system. The test item was dissolved in DMSO and tested in concentrations of 5 to 5000 µg per plate in the presence and absence of S9. In the absence of S9-mix, the test item was bacteriotoxic towards the strains TA98, TA102, and TA1537 at 1504g/plate and towards the strains TA100 and TA1535 at 5000n/plate. In the presence of S9-mix, the test item was bacteriotoxic towards all strains at 5000 µg/plate. Precipitation of the test compound on the plates was not observed. Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system. In the concentration range investigated, the test item did not induce any increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. In conclusion, these results indicate that the test item under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.


 


In vitro mammalian chromosome aberration test, RL1


This GLP compliant report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity. Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 24 hours continuous exposure in the absence of metabolic activation. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included a dose level that induced near optimum mitotic inhibition in the 4(20)-hour exposure group in the presence of S9 and in the 24 hour exposure group. Optimum toxicity was not achieved in the 4(20)-hour exposure group in the absence of S9 due to the steepness of the toxicity curve. The test material was considered to be non-clastogenic to human lymphocytes in vitro.



In vitro mammalian cell gene mutation test, RL1


This GLP compliant study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. One main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls. The exposure groups used were as follows: 4-hour exposures both with and without metabolic activation, and 24 hours without metabolic activation. The dose range of test material was selected following the results of a preliminary toxicity test and was 12.5 to 200 |jg/ml for the 4-hour exposure group in the absence of metabolic activation, 25 to 250 |jg/ml for the 4-hour exposure group in the presence of metabolic activation, and 6.25 to 150 \ig/m\ for the 24-hour exposure group in the absence of metabolic activation. The maximum dose level used was limited by test material-induced toxicity. Precipitate of test material was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in any of the three exposure groups. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.


 



Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008


The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered to be not classified for genotoxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for sixteenth time in Regulation (EU) No 2021/743.