1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +/- S9, negative, S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 (pKM101) and WP2 uvrA (pKM101), according to OECD TG 471, Sokolowski 2010

- In vitro chromosome aberration study, +S9 negative, -S9 positive, human lymphocytes, according to OECD TG 473, Bohnenberger 2010

- In vitro gene mutation assay, +/- S9 negative, mouse lymphoma L5178Y cells, according to OECD TG 476, Wollny 2010

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Mar 2010 to 28 Jun 2010

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)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Stock cultures are propagated in plastic flasks in RPMI 1640 complete culture medium. The cells are subcultured two times prior to treatment. The cell cultures are incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 week old male Wistar HsdCpb:WU rats weight approx. 220 - 320 g induced by applications of 80 mg/kg bw phenobarbital and B-naphthoflavone each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a 150 mM KCl solution (1/4, v/v) followed by centrifugation at 9000 × g. Aliquots of the supernatant were frozen and stored in ampoules at approx. -80° C. Small numbers of the ampoules were kept at -20°C for up to one week. Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo[a]pyrene. The protein concentration of the S9 preparation was 34.4 mg/mL in the pre-experiment and in experiment I, and 35.0 mg/mL in experiment II. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to give a final protein concentration of 0.75 mg/mL in the cultures. The concentration in the final test medium was 5 % (v/v).

Test concentrations with justification for top dose:

Experiment I and II:
With & without S9 mix: 420; 840; 1680; 3360; 4280 μg/mL

Vehicle / solvent:

DMSO 1 % (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

DOSING PREPARATIONS
On the day of the experiment (immediately before treatment), the test item was dissolved or suspended in DMSO. The final concentration of DMSO in the culture medium was 1 % (v/v).

EXPERIMENTAL DESIGN
The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The concentration range of the main experiments went up to approximately 10 mM. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

CELL PREPARATION
Prior to mutagenicity testing the amount of spontaneous mutants was reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with: hypoxanthine 1.0×10^-4 M, aminopterin 2.0×10^-7 M, thymidine 1.6×10^-5 M. The incubation of the cells in HAT-medium was followed by a recovery period of 2 days in RPMI 1640 medium containing: hypoxanthine 1.0×10^-4 M, thymidine 1.6×10^-5 M. After this incubation the L5178Y cells were returned to normal RPMI 1640 medium (complete culture medium). Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. Thawed stock cultures are propagated in plastic flasks in RPMI 1640 complete culture medium. The cells are subcultured two times prior to treatment. The cell cultures are incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.

CULTURE TREATMENT
In the mutation experiment 1×10^7 cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3 % horse serum were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. After 4 h the test item was removed by centrifugation and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium and incubated for an expression and growth period of 48 h. The cell density was determined each day and adjusted to 3×10^5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector. One sample of the cells was taken at the end of treatment, diluted and seeded into microtiter plates, to determine the viability of the cells after treatment (cloning efficiency 1). After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4×10^3 cells in selective medium with TFT. The viability (cloning efficiency 2) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37 ± 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated.

SIZE DISTRIBUTION OF THE COLONIES:
Colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the large colonies).

SURVIVAL
The survival rate and viability were determined based on the Poisson distribution method. The zero term of the Poisson distribution, [P(0)] method, was used. The mutation frequency was derived from the cloning efficiency under selective conditions compared to the corresponding viability under non-selective conditions.

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control. A relevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration. Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled. Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively. A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Isolated in one of the parallel cultures was noted in the first experiment at 4280 μg/mL with metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY:
No relevant cytotoxic effects indicated by a relative cloning efficiency 1 (survival) or a relative total growth (RTG) of less than 50% in both cultures occurred. An isolated cytotoxic effect in one of the parallel cultures was noted in the first experiment at 4280 μg/mL with metabolic activation. Precipitation of the test item was noted at 3360 and 4280 μg/mL in both experiments without metabolic activation. In the experimental parts with metabolic activation precipitation occurred at 1680 Sg/mL and above in the first, and at 3360 and 4280 μg/mL in the second experiment.

MUTAGENICITY:
No substantial and reproducible dose dependent increase of the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed in the main experiments with and without metabolic activation. A single, isolated increase exceeding the threshold occurred in the second culture of the second experiment without metabolic activation at a precipitating concentration of 3360 μg/mL. However, this increase was not reproduced in the parallel culture performed under identical experimental conditions. Furthermore, the increase of the mutation frequency above the threshold was not dose dependent as indicated by the lacking statistical significance and was consequently judged as biologically irrelevant artefact caused by precipitation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. No significant trend was observed in any of the experimental parts with and without metabolic activation.

In this study the range of the solvent controls was from 100 up to 196 mutant colonies per 10^6 cells; the range of the test groups treated with the test item was from 69 up to 322 mutant colonies per 10^6 cells. The highest solvent controls (196 and 188 colonies per 10^6 cells) exceeded the 50 – 170 x 10^6 control range as stated under paragraph 3.12, acceptability of the assay of this report but are within the limits recommended in reference 11. Additionally, the mutant frequency of the parallel cultures was fully acceptable (100 and 145 mutant colonies/10^6 cells). The cloning efficiency in culture II of the first experiment with metabolic activation fell just short of the lower limit of 65%. In the first culture of the second experiment without metabolic activation the cloning efficiency exceeded the upper limit of 120%. Both sets of data are acceptable however, since the cloning efficiency of the parallel cultures remained within the acceptable range.

MMS (19.5 μg/mL) and CPA (3.0 and 4.5 μg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls. In the second culture of the second experiment the positive controls with metabolic activation did not quite meet the acceptance criterion of at least 150 induced small colonies. The positive controls in the presence of metabolic activation however, were judged as valid since the positive control at 4.5 μg CPA per mL in culture II showed a substantial increase of total colonies and the corresponding
positive control of culture I easily met the acceptance criteria.

Conclusions:

In conclusion it can be stated that during the mutagenicitiy test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

An OECD 476 study was performed under GLP to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. Experiment I and II were performed with and without liver microsomal activation and a treatment period of 4 hours. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The concentrations of the test material in the Experiment I and II, with and without S9 mix were 420; 840; 1680; 3360; 4280 μg/mL.
No relevant cytotoxic effects indicated by a relative cloning efficiency 1 (survival) or a relative total growth (RTG) of less than 50% in both cultures occurred in both main
experiments with and without metabolic activation. No substantial and reproducible dose dependent increase in mutant colony numbers was observed with and without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.
In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test item is considered to be non mutagenic in this mouse lymphoma assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2010 to 28 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from two female donors (29 years old for Experiment I, 37 years old for
Experiment IIA and IIB).
Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to initiate cell cultures within 24 hrs after blood collection. If necessary, the blood was stored before use at 4 °C.
The culture medium was supplemented with 15 mM HEPES and 200 mM L-glutamine. The antibiotic solution contained 10,000 U/mL penicillin and 10,000 Ng/mL streptomycin. Additionally, the medium was supplemented with the mitogen Phytohemagglutinin , 10 % FBS (fetal bovine serum), the anticoagulant heparin.
All incubations were done at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air)

Cytokinesis block (if used):

Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital and β-naphthoflavone induced male Wistar rats. The S9 is prepared from male Wistar rats, dosed once daily for 3 consecutive days with phenobarbital (80 mg/kg bw) and β-naphthoflavone (80 mg/kg bw).
- method of preparation of S9 mix: Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment, the S9 mix was stored in an ice bath
- concentration or volume of S9 mix and S9 in the final culture medium : An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures.

Test concentrations with justification for top dose:

Experiment I, IIA, IIB -S9: 796.7, 1394.3, 2440.0, 4270.0 μg/ mL
Experiment I, IIA +S9: 455.3, 796.7, 1394.3 μg/ mL ;

Vehicle / solvent:

- Solvent used: DMSO 1.0% (v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

DOSING PREPARATIONS
Concurrent solvent controls (culture medium with 1.0 % DMSO) were performed. regarding positive controls, the dilutions of the stock solutions were prepared on the day of the experiment. Ethylmethane sulfonate was dissolved in nutrient medium, reaching final concentrations of 825.0 µg/mL (Experiment I & IIA) and 770.0 µg/mL (Experiment IIB). Cyclophosphamide was dissolved in Saline (0.9 % NaCl [w/v]), reaching a final concentration of 15.0 µg/mL (Experiment I & IIA).

EXPERIMENTAL DESIGN
Three independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment IIA, the exposure periods were 4 hours with S9 mix and 22 hours without S9 mix. In Experiment IIB, the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells per culture were counted for determination of mitotic index.

CULTURE TREATMENT
-Exposure time 4h: About 72 hrs after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes (approx. 900 x g). The supernatant with the dissolved test item was discarded and the cells were re-suspended in "saline G" (Composition per litre: NaCl 8000 mg, KCl 400 mg, glucose•H2O 1100 mg, Na2HPO4•2H20 192 mg, KH2PO4150 mg; pH was adjusted to 7.2). The washing procedure was repeated once. After washing the cells were re-suspended in complete culture medium and cultured until preparation.
-Exposure time 22h: About 72 hrs after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with complete medium (with 10% FBS) containing the test item without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed. All cultures were incubated at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

CULTURE HARVESTING
Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation (approx. 900 x g) 22 hrs after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37 °C for 20 to 25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa.

SLIDE ANALYSIS
The slides were evaluated using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates.. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 +- 1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Rationale for test conditions:

The highest concentration used in the pre-test was chosen with regard to the current OECD guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible.

The highest treatment concentration in the pre-test on toxicity (6720.0 µg/mL) was applied by mistake. Test item concentrations between 43.7 and 6720.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item was observed at the end of treatment at 2194.3 µg/mL and above. Cytotoxicity was observed after treatment with 3840.0 µg/mL in the absence of S9 mix and with 6720.0 µg/mL in the presence of S9 mix.

With regard to the molecular weight and the purity of the test item as well as the toxicity data of the pre-test, 4270.0 µg/mL (approx. 10 mM) of test item was applied as top concentration for treatment of the cultures in Experiment I. Test item concentrations between 148.7 and 4270.0 µg/mL (with and without S9 mix) were chosen. Precipitation of the test item was observed at the end of treatment at 2440.0 µg/mL and above in the absence of S9 mix and at 1394.3 µg/mL and above in the presence of S9 mix.

Using reduced mitotic indices as an indicator for toxicity in Experiment I, no clear toxic effects were observed after 4 hrs treatment in the absence and presence of S9 mix. Therefore, 4270.0 µg/mL (with and without S9 mix) was chosen as top concentration in Experiment IIA.

To verify the positive results in Experiment IIA without metabolic activation a confirmatory Experiment IIB with a top concentration of 4270.0 µg/mL was performed.

Evaluation criteria:

A test item is classified as non-mutagenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory historical control data.
-no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
-the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data and
-either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item not meeting the criteria for classification as non-mutagenic or mutagenic may be considered equivocal in this assay and may be subject to further investigation.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of the laboratory historical control data and either a concentration-related or a significant increase of the number of numerical aberrations is observed.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Species / strain:

lymphocytes: Human

Metabolic activation:

with

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In Experiment I, IIA and IIB, in the absence of S9 mix, precipitation of the test item in the culture medium was observed at 2440.0 µg/mL and above at the end of treatment. In Experiment I and IIA, in the presence of S9 mix, precipitation of the test item in the culture medium was observed at 1394.3 µg/mL and above at the end of treatment. No relevant influence or decrease in the osmolarity or pH value was observed

CYTOTOXICITY
In the absence and presence of S9 mix, no clear cytotoxicity was observed up to the highest applied concentration. However, in the absence of S9 mix in Experiment I the mitotic index was reduced to 61.1 % of control and in Experiment IIA and IIB to 68.1 and 70.7 % of control, respectively, at the highest applied concentration.

CLASTOGENIC
In Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix, no statistically significant and biologically relevant increase was observed at the concentrations evaluated. The aberration rates of the cells after treatment with the test item (0.0 – 1.5 % aberrant cells, excluding gaps) were close to the solvent control values (0.5 –3.0 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical solvent control data. In Experiment IIA in the absence of S9 mix after continuous treatment statistically significant increases at all evaluated concentrations were observed (3.0, 10.5, 4.0 % aberrant cells, excluding gaps). The values of the two highest evaluated concentrations exceeded the range of the laboratory’s historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps). In Experiment IIB in the absence of S9 mix after continuous treatment one statistically significant increase was observed at 2440.0 µg/mL (6.3 % aberrant cells, excluding gaps).
The values of the concentrations 1394.3 and 2440.0 µg/mL (3.5 and 6.3 % aberrant cells, excluding gaps) exceeded the range of the laboratory’s historical solvent control data (0.0 –3.0 % aberrant cells, excluding gaps) and thus the positive findings of Experiment IIA could be confirmed.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In the experiments, either EMS (770 or 825 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations

Table.1: Summary of results of the chromosomal aberration study

Exp.	Preparation interval	Test item concentration in µg/mL	Mitotic indices in % of control	Incl. gaps*	Aberrant cells in % excl. gaps*	carrying exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1	100.0	1.0	0.5	0.0
		Positive control^2	78.4	12.0	12.0^S	3.0
		1394.3	97.3	0.5	0.5	0.0
		2440.0^P	74.4	0.0	0.0	0.0
		4270.0^P	61.1	1.0	1.0	0.0
Exposure period 22 hrs without S9 mix
IIA	22 hrs	Solvent control^1	100.0	0.5	0.5	0.0
		Positive control^2	49.9	23.0	22.5^S	6.5
		1394.3^#	96.7	4.0	3.0^S	0.3
		2440.0^P	77.3	11.5	10.5^S	0.0
		4270.0^P	68.1	7.0	4.0^S	0.0
IIB	22 hrs	Solvent control^1	100.0	2.0	2.0	0.0
		Positive control^3	65.9	16.0	14.0^S	4.5
		796.7	81.5	2.0	1.5	0.0
		1394.3^#	72.7	3.5	3.5	0.0
		2440.0^#P	68.3	6.8	6.3^S	0.0
		4270.0^#P	70.7	3.8	3.0	0.0
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1	100.0	1.0	1.0	0.0
		Positive control^2	57.9	12.5	12.0^S	3.0
		455.3	99.4	1.0	1.0	0.0
		796.7	84.4	1.0	1.0	0.0
		1394.3^P	95.3	0.5	0.5	0.0
IIA	22 hrs	Solvent control^1	100.0	3.0	3.0	0.0
		Positive control^2	57.5	18.0	17.5^S	3.0
		455.3	85.5	0.5	0.5	0.0
		796.7	91.3	1.0	1.0	0.0
		1394.3^P	90.8	2.0	1.5	0.0

* Including cells carrying exchanges
P Precipitation occurred at the end of treatment
S Aberration frequency statistically significant higher than corresponding control values
1 DMSO 1.0 % (v/v)
2 CPA 15.0 µg/mL

Conclusions:

It can be stated that under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro in the absence of metabolic activation, when tested up to the highest required concentration.

Executive summary:

This in vitro OECD 473 genotoxicity assay was performed under GLP to assess the potential of the test item to induce structural chromosomal aberrations in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/b-naphthoflavone treated male rats) at concentration of Experiment I; 1394.3, 2440.0, 4270.0 µg/mL and 455.3, 796.7, 1394.3 µg/mL in the absence and presence of metabolic activation (S9-mix);  and Experiment IIA 1394.3, 2440.0, 4270.0 µg/mL and 455.3, 796.7, 1394.3 µg/mL in the absence and presence of S9-mix and Experiment IIB 796.7, 1394.3, 2440.0, 4270.0 µg/mL in the presence of S9-mix.

In each experimental group two parallel cultures were analysed.  Per culture at least 100 metaphases were scored for structural chromosomal aberrations.  The highest applied concentration in the main study (4270.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity of the test item and with respect to the current OECD Guideline 473.  Dose selection of the cytogenetic experiments was performed considering the toxicity data and test item precipitation and in accordance with OECD Guideline 473. 

In the absence and presence of S9 mix, no clear cytotoxicity was observed up to the highest applied concentration.  However, in the absence of S9 mix in Experiment I the mitotic index was reduced to 61.1 % of control and in Experiment IIA and IIB to 68.1 and 70.7 % of control, respectively, at the highest applied concentration.  In Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix, no clastogenicity was observed at the concentrations evaluated.  In Experiment IIA in the absence of S9 mix after continuous treatment statistically significant increases at all evaluated concentrations were observed (3.0, 10.5, 4.0 % aberrant cells, excluding gaps).  The values of the two highest concentrations exceeded the range of the laboratory’s historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps).  In Experiment IIB in the absence of S9 mix after continuous treatment one statistically significant increase was observed at 2440.0 µg/mL (6.3 % aberrant cells, excluding gaps). The values of the concentrations 1394.3 and 2440.0 µg/mL (3.5 and 6.3 % aberrant cells, excluding gaps) exceeded the range of the laboratory’s historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and thus the positive findings of Experiment IIA could be confirmed.  

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.  Appropriate mutagens were used as positive controls.  They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro.  Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence of metabolic activation, when tested up to the highest required concentration.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Apr 2010 to 25 May 2010

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium) and trp- (E.coli) strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

(uvrA pKM 101 and pKM 101)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: phenobarbital and β-naphthoflavone induced rat liver
- Concentration or volume of S9 mix and S9 in the final culture medium: The S9 is prepared from male rats (Sprague-Dawley) induced with 80 mg/kg bw phenobarbital and 80 mg/kg bw β-naphthoflavone. The protein concentration in the S9 preparation was 34.3 mg/mL. S9 supernatant was thawed and mixed with S9 cofactor solution (8mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4)
- Quality controls of S9: Positive control substances are tested to confirm the activity of the S9-mix.

Test concentrations with justification for top dose:

Experiment I (pre-experiment): 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate;
Experiment II:
-Salmonella strains:1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
-E. coli strains: 33; 100; 333; 1000; 2500; and 5000 µg/plate;
Experiment IIa: 0.03; 0.1; 0.3; 1; 3; 33; 100 µg/plate

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

EXPERIMENTAL PERFORMANCE
To evaluate the toxicity of the test item a pre-experiment was performed with all strains. Eight concentrations were tested for toxicity and mutation induction each with three replicate plates. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, if the following criteria are met: A minimum of five analysable dose levels should be present with at least four dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.The above criteria should be met for all valid experiments. In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. Since toxic effects were observed in the Salmonella strains nine concentrations were tested in the Salmonella strains and 6 concentrations were chosen for the E.coli strains in experiment II (pre-incubation test) and 5000 µg/plate was chosen as maximal concentration. Due to strong toxic effects in strain TA 1537 in experiment II in the absence of metabolic activation, this part was repeated with lower concentrations (reported as experiment IIa).
The following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control), 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer* (for test without metabolic activation), 100 µL Bacteria suspension (cf. test system, pre-culture of the strains), 2000 µL Overlay agar.

In the pre-incubation assay 100 µL test solution (solvent control), 100 µL reference mutagen solution (positive control), 500 µL S9 mix / S9 mix substitution buffer* and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre- incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark
For each strain and dose level, including the controls three plates were used.
The colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager

Rationale for test conditions:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.

Key result

Species / strain:

E. coli WP2

Remarks:

pKM 101

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

-No reduced background growth was observed in the experiments with and without metabolic activation. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at certain concentrations (µg/plate) (Table 2)
-No precipitation (visible to the unaided eye) of the test item was observed either in the test tubes or on the incubated agar plates.
-No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
-Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Table 2. Toxic effects concentrations (µg/plate) observed in the test groups

 

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	1000 - 5000	/	1000 - 5000	2500 - 5000
TA 1537	333 - 5000	2500 - 5000	33, 1000 - 5000	1000 - 5000
TA 98	1000 - 5000	1000 - 5000	1000 - 5000	2500 - 5000
TA 100	2500 - 5000	5000	2500 - 5000	2500 - 5000
WP2 uvrA pKM 101	/	/	/	/
WP2 pKM 101	/	/	/	/

/ = no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)

Conclusions:

During the described mutagenicity tests and under the experimental conditions reported,
the test item did not induce gene mutations by base pair changes or frame shifts in the genome
of the strains used.
The test item is considered to be non-mutagenic in the Salmonella typhimurium and
Escherichia coli reverse mutation assay

Executive summary:

An OECD TG 471 study was performed under GLP to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2 uvrA pKM 101 and WP2 pKM 101 over the concentration range, Experiment I:  3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate and Experiment II: for Salmonella strains:  1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate and for E. coli strains:  33; 100; 333; 1000; 2500; and 5000 µg/plate in the absence and presence of metabolic activation (S9-mix) and in Experiment IIa:  0.03; 0.1; 0.3; 1; 3; 33; 100 µg/plate in the absence of S9-mix only.

No reduced background growth was observed with and without metabolic activation in all strains used in both experiments.  Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in all Salmonella strains.

No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).  There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.  Positive control chemicals showed appropriate responses in the relevant strains.

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus test in mice, negative, according to OECD TG 474, Merker 2010

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:

07 Sep 2010 to 06 Oct 2010

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

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 – 9 weeks
- Weight at study initiation: mean value 34.4g (*SD ± 1.8g); 31.1 – 37.9 g
- Housing: single; in Makrolon Type II/III, with wire mesh top
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water. Ad libitum
- Acclimatisation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 45 - 71 %
- Photoperiod: Artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: 09 September 2010 to: 06 October 2010

Route of administration:

oral: gavage

Vehicle:

sterile water

Details on exposure:

FORMULATION:
On the day of the experiment, the test item was formulated in sterile water
Volume administered: 10 mL/kg body weight.

Duration of treatment / exposure:

Bone marrow samples were collected at the central sampling interval of 24 h after treatment.
For the highest dose level an additional bone marrow sample was taken at 48 h after treatment

Frequency of treatment:

Animals received a single dose of the test item

Post exposure period:

The animals of all dose groups, except the positive control were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h, 24 h, and 48 h after administration of the test item or the vehicle controls. Sampling of the bone marrow was done 24 and 48 hours after treatment

Dose / conc.:

500 other: mg/kg bw

Remarks:

24 h preparation interval

Dose / conc.:

1 000 other: mg/kg bw

Remarks:

24 h preparation interval

Dose / conc.:

2 000 other: mg/kg bw

Remarks:

24 h preparation interval

Dose / conc.:

2 000 other: mg/kg bw

Remarks:

48 h preparation interval

No. of animals per sex per dose:

14 males were assigned to the high dose group
7 males were assigned to the middle dose group
7 males were assigned to the low dose group
5 males were assigned to negative control groups
5 males were assigned to positive control groups

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide:
- Route of administration: oral
- Dose: 40 mg/kg b.w., once
-Volume: 10 mL/kg b.w.

Tissues and cell types examined:

Bone marrow; 2000 polychromatic erythrocytes (PCE) per animal were analysed for
micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed in both male and female mice (two animals per sex per dose level) under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration. The animals were treated once orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item. The test dose levels were chosen according to the following scheme: 5 – 8 – 12.5 – 20 – 32 – 50 – 80 – 125 – 200 – 320 – 500 – 800 – 1250 – 2000 mg/kg b.w.. No substantial sex specific differences on toxic symptoms were observed, therefore, the main experiment was performed using male animals only. The treated animals did not express any clinical signs of toxicity. On the basis of these data 2000 mg/kg b.w., the maximum OECD Guideline recommended dose for this assay, was considered suitable. No gender specific differences in toxicity were observed, thus, the main study was performed using male animals only, as permitted by the Guideline.

TREATMENT AND SAMPLING TIMES:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test item, the vehicle or the positive control substance once orally. Seven males were treated per dose group and sampling time. Five males each were treated for the vehicle and positive control group. The animals of all dose groups, except the positive control were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h, 24 h, and 48 h after administration of the test item or the vehicle controls. Sampling of the bone marrow was done 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 × g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100× oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Immature and mature erythrocytes were identified by their pale and blue to green colour, respectively. Micronuclei are distinguished by being small nuclei separate from and additional to the main nuclei of the cells.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear
increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
Statistical methods were used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of
micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
A test item failing to meet the criteria for a positive or negative response may be judged
equivocal in this assay and may be considered for further investigation.

ACCEPTANCE CRITERIA:
The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase
of micronucleated PCEs compared to the negative control.

Statistics:

Statistical methods were used as an aid in evaluating
the results: Statistical significance at the five per cent level (p < 0.05) for the incidence of micronuclei was evaluated by means of the non-parametric Mann-Whitney test

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals did not express any toxic reactions. The animals of the vehicle control groups (sterile water) for both sampling times did also not express any toxic reactions.
The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control, indicating that the test substance did not have any cytotoxic properties in the bone marrow.
In comparison to the corresponding vehicle controls there was no biologically relevant enhancement and statistically significant increase in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item.
The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the respective vehicle control group and mainly within the historical vehicle control range. Additionally no clear dose dependence could be observed and furthermore the group of animals sampled 48 hours after treatment with the highest test item dose level did not show any statistically or biologically relevant enhancement of micronucleated PCEs.
A dose of 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control, which showed a substantial increase of induced micronucleus frequency.  The volume of the positive control administered was 10 mL/kg b.w.

Table 1. Summary of Micronucleus Test Results

test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythrocytes
vehicle	0	24	0.130	1 -4	1227
test item	500	24	0.086	0 -4	1220
test item	1000	24	0.071	0 -4	1172
test item	2000	24	0.086	0 -5	1217
positive control	40	24	2.760	42 -67	1068
vehicle	0	48	0.070	0 -3	1216
test item	2000	48	0.100	0 -4	1215

Conclusions:

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Therefore, the test item is considered to be non-mutagenic in this bone marrow micronucleus assay.

Executive summary:

An OECD TG 474 genetic toxicity study was performed under GLP in order to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (NMRI).  The test item was formulated in sterile water, which was also used as vehicle control.  The volume administered orally was 10 mL/kg body weight (b.w.).  At 24 h and 48 h after a single administration of the test item, the bone marrow cells were collected for micronuclei analysis.

Seven males per test group (except the control groups with 5 males only) were evaluated for the occurrence of micronuclei.  Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.  To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.  The following dose levels of the test item were investigated:  24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.  48 h preparation interval: 2000 mg/kg b.w.  The highest dose was estimated by a pre-experiment to be suitable. 

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control, thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.  In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.  The mean values of micronuclei observed after treatment with the test item were below or near to the value of the vehicle control group. 

A dose of 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control, which showed a substantial increase of induced micronucleus frequency.  The volume of the positive control administered was 10 mL/kg b.w.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.  Therefore, the test item is considered to be non-mutagenic in this bone marrow micronucleus assay. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

All available data was assessed and the studies representing the worst-case effects were included as key studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA. One in vitro chromosome aberration study in mammalian cells (Bohnenberg 2010) showed a positive result for structural chromosomal aberrations in human lymphocytes in the absence of metabolic activation. Clastogenicity was further evaluated in vitro (Hertner 1993) and in vivo (Strasser 1987). Both studies did not show any evidence of clastogenic effects and no evidence of toxic or mutagenic effects on somatic interphase cells, it was therefore concluded that the test item is non-genotoxic.

Multiple genotoxicity studies with metabolites of the test substance are available, of which non indicated a genotoxic potential. In total, there are 5 Ames tests, 5 clastogenicity tests, 4 mammalian gene mutation tests and 2 DNA repair tests. In addition, one in vivo micronucleus test in Chinese hamsters is available.

 

Genetic toxicity in vitro
Ames
The test material was evaluated in a bacterial reverse mutation assay using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two strains of Escherichia coli (WP2 (pKM101) and WP2 uvrA (pKM101). This study was conducted in accordance with OECD TG 471 following GLP principles (Sokolowski 2010). The investigations were performed with concentrations of 1; 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate for the S. typhimurium and in concentrations of 33; 100; 333; 1000; 2500; and 5000 µg/plate; for the E. Coli strains. Due to strong toxic effects in strain TA 1537 in the absence of metabolic activation, this part was repeated with concentrations of 0.03; 0.1; 0.3; 1; 3; 33; 100 µg/plate. The experiments were conducted in the presence and absence of a rat liver-derived metabolic activation system (S9 mix).
The test material did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the strains used, either in the presence or absence of S9 mix. The sensitivity of the test system, and the metabolic activity of the S9 mix, were clearly demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.
The test material gave a negative (non-mutagenic) response in both the presence and absence of S9 mix.

In vitro chromosome aberration study in mammalian cells
The test material was evaluated for its clastogenic potential in an in vitro cytogenetic assay using human lymphocytes in two independent experiments treated in the presence and absence of a rat liver-derived metabolic activation system (S9 mix) (Bohnenberg 2010). This study was conducted in accordance with OECD TG 473 following GLP guidelines. In Experiment 1, cultures were treated for a period of 4 hours both in the presence and absence of S9 mix. In Experiment 2a, cultures were treated for a period of 4 hours in the presence of S9 mix and 22 hours in the absence of S9 mix. In Experiment 2b, the exposure period was 22 hours without S9 to verify the positive results in experiment 2a.
All cultures were harvested 22 hours after the beginning of treatment. The concentrations were chosen to be 148.7, 260.2, 455.3, 796.7, 1394.3, 2440.0, 4270.0 μg/ mL (+S9 -mix); 148.7, 260.2, 455.3, 796.7, 1394.3, 2440.0, 4270.0 μg/ mL (-S9 -mix) in Experiment 1, and 260.2, 455.3, 796.7, 1394.3, 2440.0, 4270.0 μg/ mL (+S9 -mix) and 27.7, 48.5, 84.9, 148.7, 260.2, 455.3, 796.7, 1394.3, 2440.0, 4270.0 μg/ mL (-S9 -mix) in Experiment 2a and 148.7, 260.2, 455.3, 796.7, 1394.3, 2440.0, 4270.0 μg/ mL (-S9 -mix) in Experiment 2b. Cultures were treated with the test material at appropriate concentrations for chromosomal aberration analysis along with the appropriate solvent and positive control cultures. The highest concentrations selected for chromosome aberration analysis was chosen with regard to the molecular weight and the purity of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In the absence and presence of S9 mix, no clear cytotoxicity was observed up to the highest applied concentration. However, in the absence of S9 mix in Experiment 1, the mitotic index was reduced to 61.1 % of control and in Experiment 2a and 2b to 68.1 and 70.7 % of control, respectively, at the highest applied concentration. Moreover, In Experiment 1 in the absence and presence of S9 mix and in Experiment 2A in the presence of S9 mix, no statistically significant and biologically relevant increase was observed at the concentrations evaluated. The aberration rates of the cells after treatment with the test item (0.0 – 1.5 % aberrant cells, excluding gaps) were close to the solvent control values (0.5 –3.0 % aberrant cells, excluding gaps) and within the range of the laboratory’s historical solvent control data. In Experiment 2a in the absence of S9 mix after continuous treatment statistically significant increases at all evaluated concentrations were observed (3.0, 10.5, 4.0 % aberrant cells, excluding gaps). The values of the two highest evaluated concentrations exceeded the range of the laboratory’s historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps). In Experiment 2b in the absence of S9 mix after continuous treatment one statistically significant increase was observed at 2440.0 µg/mL (6.3 % aberrant cells, excluding gaps). The values of the concentrations 1394.3 and 2440.0 µg/mL (3.5 and 6.3 % aberrant cells, excluding gaps) exceeded the range of the laboratory’s historical solvent control data (0.0 –3.0 % aberrant cells, excluding gaps) and thus the positive findings of Experiment 2a could be confirmed.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Therefore, the test material induces structural chromosomal aberrations in human lymphocytes in vitro in the absence of metabolic activation, when tested up to the highest required concentration.

The other in vitro chromosome aberration study in mammalian cells, also performed according to OECD TG 473 and GLP, is a supporting study (Hertner 1993). In this study, no clastogenic effects were observed in Chinese hamster ovary cells exposed to concentrations up to 250 µg/mL in presence of S9 mix or 16.0 µg/mL in absence of S9 mix.

In vitro gene mutation assay
Three supporting studies to assess the potential of the test material to cause gene mutation effects were performed. A cell mutation assay at the Thymidine Kinase Locus (TK+/-) in mouse lymphoma L5178Y cells was conducted in accordance with OECD TG 476 following GLP principles (Wollny 2010). In this study, the test material resulted not mutagenic in L5178Y TK+/- cells treated in vitro in either the presence or absence of S9 mix. Another mouse lymphoma L5178Y (TK+/-) assay (Dollenmaier 1986) concluded that the test item showed no evidence of mutagenic effects in either the presence or absence of S9 mix. A mutagenicity test in Saccharomyces Cerevisiae D7 (Hool 1984) concluded that the test item did not show evidence of mutagenic effects.

In vitro DNA damage and/or repair study
Two supporting studies were conducted to assess the potential of the test item or its metabolites to induce DNA damage that could be interpreted as suggestive of mutagenic or carcinogenic properties. The first study was performed on rat hepatocytes in vitro, following GLP principles (Puri, 1984). It was concluded that no evidence of induction of DNA damage by the test item or by its metabolites was observed. The second study was performed on human fibro blasts in vitro, following GLP principles (Puri, 1984). It was concluded that no evidence of induction of DNA damage by the test item was obtained that could be interpreted as suggestive of mutagenic or carcinogenic properties of the substance.

In vitro transformation study
The test item was tested in a GLP supporting study for transformation inducing effects on mouse fibroblasts (BALB/3T3) without and with metabolic activation (Meyer 1986). It was concluded that no effects were obtained that must be interpreted as suggestive of a transformative property of the test item and its metabolites.

Genetic toxicity in vivo
In vivo micronucleus test
The test material has been evaluated for its ability to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse in a study according to OECD TG 474 following GLP principles (Merker 2010). A single oral dose was given to groups of male mice at dose levels of 500, 1000 and 2000 mg/kg. Bone marrow samples were taken 24 and 48 hours after dosing. No significant increases in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. Comparison of the percentage of immature erythrocytes showed no significant differences at either of the sampling times between the vehicle control animals and those treated with the test material. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the respective vehicle control group and mainly within the historical vehicle control range. Cyclophosphamide administered orally was used as positive control, which showed a substantial increase of induced micronucleus frequency, thus demonstrating the sensitivity of the test system to a known clastogen.
Under the conditions of test, the test material is considered to be non-mutagenic.

Other two supporting studies were performed to evaluate the genotoxicity of the test item in vivo. One cytogenicity, bone marrow chromosome aberration test (Strasser 1987) was performed in Chinese hamsters to evaluate any toxic and mutagenic effect on somatic interphase cells, resulting in no evidence of toxic or mutagenic effects. A mammalian spot test (Strasser 1986) was performed in mice to ascertain whether the test substance has a mutagenic effect on somatic cells in vivo resulting in no evidence of mutagenic effects obtained in the offspring of pregnant mice.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.