4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames, +S9 negative, -S9 negative, S. typhimurium: TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA pKM101 and WP2 pKM101, OECD 471, Chang 2016 and 2019

- in vitro chromosome aberration test, +S9 negative, -S9 negative, human lymphocytes, OECD 473, Chang 2019

- in vitro mammalian cell gene mutation test (MLA), +S9 negative, -S9 negative, L5178Y mouse lymphoma cells, OECD 490, Sokolowski 2019

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Mar 2019 to 08 Apr 2019

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:

July 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium), trp- (E. coli)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

E. coli, other: WP2 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/ß-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: The S9 was prepared from male Wistar rats (RjHan:WI; weight approx. 220 – 320 g) induced by peroral administration of 80 mg/kg b. phenobarbital and by peroral administrations of ß-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 KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. The protein concentration in the S9 preparation was 34.4 mg/mL in the pre-experiment / Experiment I and 33.3 mg/mL in experiment II. Experiment IIa was performed without S9 mix only.
- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10 % v/v in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM 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 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.

Test concentrations with justification for top dose:

3; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

PRE-EXPERIMENT FOR CYTOTOXICITY
To evaluate the cytotoxicity of the test substance a pre-experiment was performed with all strains. Eight concentrations were tested for cytotoxicity and mutation induction each with three replicate plates. The experimental conditions in this pre-experiment were the same as described below for experiment I (plate incorporation test). Cytotoxicity of the test substance results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn. The pre-experiment is reported as the main experiment I since the criteria mentioned under Acceptability of the Assay were met.

EXPERIMENTAL PERFORMANCE
For each strain and concentration including the controls, three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each concentration, 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; OD = 0.9 - 1.2; wavelength = 500 nm; approx. 8x108 cells/mL),
2000 µL Overlay agar
For the pre-incubation method test solution (100 µL) (solvent or reference mutagen solution (positive control)), S9 mix / S9 mix substitution buffer* (500 µL) and bacteria suspension (100 µL) were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre- incubation overlay agar (2.0 mL, 45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark, plates were then stored at 4 °C until counted.
In parallel to each test a sterile control of the test substance was performed and documented in the raw data. Therefore, stock solution (100 μL) and S9 mix / S9 mix substitution buffer* (500 µL) were mixed with overlay agar (2.0 mL) and poured on minimal agar plates.
* Substitution buffer: 7 parts of the 100 mM sodium-ortho-phosphate-buffer pH 7.4 with 3 parts of KCl solution 0.15 M.

DATA RECORDING
The colonies were counted using a Petri Viewer with the software program Ames Study Manager. The evaluation unit was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). The print outs are kept with the raw data. Due precipitation of the test substance some test groups were scored manually.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
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 the historical data
- the positive control substances should produce an increase in mutant colony frequencies of at least 2-fold concurrent control
- a minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

EVALUATION OF RESULTS
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants of twice or above the spontaneous mutation rate of the corresponding solvent control is observed.
A concentration dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration (6).
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration 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. However, whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

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 applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli, other: WP2 uvrA pKM101 and WP2 pKM101

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES
In the pre-experiment the concentration range of the test substance was 3 - 5000 µg/plate. The pre-experiment is reported as Experiment I. Since no relevant cytotoxic effects were observed 5000 µg/plate was chosen as the maximal concentration in Experiment II. Since no bacterial colony growth occurred in the positive control of strain TA1535 without S9 mix in Experiment II, this part was repeated under the same conditions as Experiment II (reported as Experiment IIa). Experiments I and II were performed with and without liver microsomal activation, Experiment IIa only without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The concentration range included two logarithmic decades. The test substance was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
Experiments II and IIa: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

STUDY RESULTS
Ames test:
- Signs of toxicity: The plates incubated with the test substance showed normal background growth up to the maximal dose of 5000 µg/plate with and without S9 mix in all strains used. Minor cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in Experiment I in strain WP2 uvrA (pKM101) at 5000 µg/plate with and without S9 mix. No further cytotoxic effects were observed, neither in the remaining test groups of Experiment I, nor in Experiments II and IIa.
- Mean number of revertant colonies per plate and standard deviation: No relevant increase in revertant colony numbers in any of the six tester strains was observed following treatment with test substance at any concentration 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 in- crease in induced revertant colonies.

Table 1. Summary of Results Pre-Experiment/Experiment I

Metabolic Activation	Test Group	Concen tration (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO		9 ± 1	8 ± 3	21 ± 6	111 ± 17	263 ± 19	318 ± 83
	Untreated		13 ± 3	13 ± 7	24 ± 8	125 ± 12	276 ± 32	282 ± 5
	test substance	3 µg	10 ± 5	11 ± 4	25 ± 8	102 ± 6	227 ± 20	370 ± 54
	.	10 µg	12 ± 4	9 ± 2	25 ± 7	132 ± 5	249 ± 7	274 ± 29
		33 µg	9 ± 0	12 ± 1	23 ± 3	120 ± 13	246 ± 5	237 ± 7
		100 µg	9 ± 4	9 ± 3	23 ± 3	122 ± 15	248 ± 19	198 ± 16
		333 µg	12 ± 2^P	11 ± 1^P	22 ± 5^P	112 ± 26^P	246 ± 8^P	234 ± 16^P
		1000 µg	9 ± 2^PM	6 ± 3^PM	23 ± 4^PM	130 ± 16^P	232 ± 4^P	148 ± 2^PM
		2500 µg	10 ± 2^PM	7 ± 2^PM	17 ± 6^PM	86 ± 7^PM	159 ± 20^PM	154 ± 9^PM
		5000 µg	7 ± 2^PM	7 ± 2^PM	14 ± 3^PM	86 ± 2^PM	134 ± 25^PM	126 ± 14^PM
	NaN3	10 µg	927 ± 29			1679 ± 73
	4-NOPD	10 µg			449 ± 40
	4-NOPD	50 µg		68 ± 10
	MMS	2.0 µL					3641 ± 308	3096 ± 193
With Activation	DMSO		10 ± 4	11 ± 3	40 ± 7	125 ± 27	280 ± 8	364 ± 76
	Untreated		10 ± 1	14 ± 7	42 ± 7	131 ± 11	300 ± 33	375 ± 51
	test substance	3 µg	12 ± 4	11 ± 6	42 ± 6	119 ± 7	289 ± 36	505 ± 53
		10 µg	9 ± 3	10 ± 1	46 ± 5	114 ± 13	281 ± 53	479 ± 113
		33 µg	10 ± 4	11 ± 5	48 ± 6	139 ± 12	278 ± 19	264 ± 26
		100 µg	10 ± 4	12 ± 4	33 ± 6	122 ± 4	280 ± 25	255 ± 13
		333 µg	9 ± 3^P	10 ± 3^P	41 ± 6^P	133 ± 11^P	261 ± 11^P	453 ± 22^P
		1000 µg	10 ± 3^PM	9 ± 1^PM	27 ± 2^PM	117 ± 7^P	243 ± 34^P	263 ± 10^PM
		2500 µg	10 ± 2^PM	9 ± 3^PM	33 ± 4^PM	87 ± 8^PM	232 ± 22^PM	224 ± 8^PM
		5000 µg	7 ± 2^PM	7 ± 3^PM	26 ± 5^PM	91 ± 10^PM	183 ± 30^PM	153 ± 17^PM
	2-AA	2.5 µg	423 ± 17	233 ± 45	3416 ± 163	3824 ± 210
	2-AA	10.0 µg					1105 ± 106	1945 ± 223

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3	sodium azide	P	Precipitate
2-AA	2-aminoanthracene	M	Manual count
4-NOPD	4-nitro-o-phenylene-diamine
MMS	methyl methane sulfonate

Table 2: Summary of Results Experiment II

Metabolic Activation	Test Group	Concen tration  (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO		n.r.*	13 ± 2	24 ± 7	110 ± 5	277 ± 6	299 ± 15
	Untreated		n.r.*	14 ± 4	27 ± 4	132 ± 14	289 ± 20	343 ± 10
	test substance	33 µg	n.r.*	11 ± 1	25 ± 5	113 ± 14	262 ± 16	324 ± 28
		100 µg	n.r.*	12 ± 2	24 ± 7	116 ± 26	248 ± 25	302 ± 34
		333 µg	n.r.*	14 ± 2^P	27 ± 6^P	116 ± 8^P	251 ± 17^P	270 ± 13^P
		1000 µg	n.r.*	8 ± 2^P	19 ± 1^PM	106 ± 5^PM	243 ± 3^P	285 ± 14^PM
		2500 µg	n.r.*	7 ± 2^PM	17 ± 3^PM	81 ± 9^PM	184 ± 11^PM	261 ± 4^PM
		5000 µg	n.r.*	7 ± 1^PM	14 ± 4^PM	81 ± 16^PM	143 ± 9^PM	183 ± 6^PM
	NaN3	10 µg	Not valid			1821 ± 30
	4-NOPD	10 µg			450 ± 15
	4-NOPD	50 µg		87 ± 14
	MMS	2.0 µL					3558 ± 89	3185 ± 107
With Activation	DMSO		10 ± 4	11 ± 2	34 ± 8	158 ± 26	294 ± 25	365 ± 27
	Untreated		13 ± 2	13 ± 2	41 ± 6	139 ± 21	334 ± 18	387 ± 20
	test substance	33 µg	11 ± 4	10 ± 2	31 ± 7	143 ± 2	307 ± 25	390 ± 32
		100 µg	12 ± 0	9 ± 2	35 ± 6	142 ± 16	283 ± 16	341 ± 11
		333 µg	11 ± 2^P	10 ± 2^P	31 ± 6^P	123 ± 13^P	293 ± 14^P	309 ± 18^P
		1000 µg	10 ± 0^P	9 ± 3^PM	37 ± 5^P	125 ± 1^P	275 ± 22^P	434 ± 11^PM
		2500 µg	8 ± 1^PM	8 ± 2^PM	24 ± 7^PM	129 ± 5^PM	238 ± 13^PM	348 ± 11^PM
		5000 µg	6 ± 2^PM	6 ± 2^PM	23 ± 5^PM	117 ± 6^PM	188 ± 18^PM	272 ± 26^PM
	2-AA	2.5 µg	436 ± 21	112 ± 11	3389 ± 446	4087 ± 331
	2-AA	10.0 µg					976 ± 49	1820 ± 30

  

Key to Positive Controls		Key to Plate Postfix Codes
NaN3	sodium azide	P	Precipitate
2-AA	2-aminoanthracene	M	Manual count
4-NOPD	4-nitro-o-phenylene-diamine
MMS	methyl methane sulfonate

Conclusions:

In conclusion, it can be stated that during the described mutagenicity tests and under the experimental conditions reported, test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test sunstance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This GLP compliant OECD 471 study was performed to investigate the potential of test substance to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II and IIa) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA (pKM101) and WP2 (pKM101).

The plates incubated with the test item showed normal background growth up to the maximal dose of 5000 µg/plate with and without S9 mix in all strains used. Minor cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in Experiment I in strain WP2 uvrA (pKM101) at 5000 µg/plate with and without S9 mix. No further cytotoxic effects were observed, neither in the remaining test groups of Experiment I, nor in Experiments II and IIa. No relevant increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).  There was also no observed 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, which showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity tests and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation 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:

06 Jan 2016 to 08 Mar 2016

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:

September 2014

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human lymphocytes

For lymphocytes:
- Sex, age and number of blood donors: Blood samples were obtained from one healthy non-smoking male donor (32 years old) not receiving medication. Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to performing laboratory to initiate cell cultures within 24 h after blood collection Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohemagglutinin to the culture medium for a period of 48 hours. The cell harvest time point was approximately 1.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Culture conditions
- Blood cultures were established by preparing an 11% mixture of whole blood in medium within 30 h after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (phytohemagglutinin) (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
The following volumes were added to the flasks (per 10 mL): 7.60 mL culture medium; 1.00 mL fetal bovine serum; 0.10 mL antibiotic solution; 0.05 mL phytohemagglutinin; 0.05 mL heparin; 0.10 mL HEPES; 1.10 mL whole blood
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Liver S9 obtained from phenobarbital/-naphthoflavone induced rats was used as the metabolic activation system.
- Method of preparation of S9 mix: The S9 was prepared from 8 - 14 weeks old male Wistar rats, treated by oral administration on three days (24 ± 4 h) with Phenobarbital and Beta-naphthoflavone (each 80 mg/kg body weight per day). 24 ± 4 h after the last treatment the the S9 was prepared and stored. Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
The protein concentration of the S9 preparation was 27.2 mg/mL.
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. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium- ortho-phosphate-buffer (100 mM, pH 7.4).

Test concentrations with justification for top dose:

- Pre-test (Experiment I): 4.3, 7.5, 13.2, 23.0 µg/mL (-S9, 4-hour exposure)
- Experiment II: 6.4, 11.2, 19.6, 34.4 µg/mL (-S9, 22-hour exposure)
- Pre-test (Experiment I): 7.5, 23.0, 40.3, 80.6 µg/mL (+S9, 4-hour exposure)

Vehicle / solvent:

- Solvent used: DMSO (1.0% (v/v) in culture medium)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

RANGE-FINDER:
A pre-experiment was performed to evaluate the cytotoxicity of test substance. The pre- experiment is reported as the main experiment I since the criteria mentioned under Acceptability of the assay were met.
With regard to the purity (96.6%) of the test substance, 5160.0 µg/mL was applied as the top concentration for treatment of the cultures in Experiment I. Test substance concentrations ranging from 4.3 to 5160.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test substance was observed at the end of treatment at 23.0 µg/mL and above in the absence of S9 mix and at 80.6 µg/mL and above in the presence of S9 mix.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, moderate toxic effects were observed after 4 hours treatment with 80.6 µg/mL and above in the absence of S9 mix and with 161.3 µg/mL and above in the presence of S9 mix. Therefore, 322.5 µg/mL (without S9 mix) were chosen as top concentration in Experiment II. Since the cytotoxicity for the positive controls exceeded the acceptability criteria in Experiment II, the experiment was repeated using the same concentration range.

TREATMENT EXPOSURE TIME 4 HOURS:
About 48 h 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 substance.
For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 h the cells were spun down by gentle centrifugation for 5 minutes (approx. 900 x g). The supernatant with the dissolved test substance was discarded and the cells were re-suspended in "saline G". The washing procedure was repeated once as described.
The "saline G" solution was composed as follows (per litre): 8000 mg NaCl, 400 mg KCl, 1100 mg glucose.H2O, 192 mg Na2HPO4.2H20, 150 mg KH2PO4, with the pH adjusted to 7.2.
After washing the cells were re-suspended in complete culture medium and cultured until preparation.

EXPOSURE TIME 22 HOURS (WITHOUT S9 MIX):
About 48 h 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 substance 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).

PREPARATION OF THE CULTURES:
The cultures were treated with the metaphase-arresting substance Colcemid (final concentration: 0.2 µg/mL) approximately three hours before the requested harvest time. The cultures were harvested by centrifugation 22 h after beginning of treatment. The supernatant was discarded and the cells were resuspended in hypotonic solution (0.0375 M KCl). Then the cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation (approx. 900 x g) the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

ANALYSIS OF METAPHASE CELLS
The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") 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. 150 well-spread metaphases per culture (total 300) were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 2 centromere regions were included in the analysis. To describe a cytotoxic effect, the mitotic index (% cells in mitosis) was determined.

DATA RECORDING
The generated data were recorded in the laboratory documentation. The results were presented in tabular form, including experimental groups with the test substance, solvent controls, and positive controls, respectively.

Evaluation criteria:

See 'Any other information on materials and methods incl. tables'.

Statistics:

Data were evaluated for statistical significance using the Fisher Exact Probability Test (one-sided) (p < 0.05).

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRECIPITATION:
Experiment I: precipitation of the test substance in the culture medium was observed in the absence of S9 mix at 23.0 µg/mL and above and in the presence of S9 mix at 80.6 µg/mL and above at the end of treatment.
Experiment II: in the absence of S9 mix after continuous treatment precipitation was observed at 60.2 µg/mL and above.

OSMOLARITY AND PH:
The osmolarity and pH were determined in the solvent control and the maximum concentration without metabolic activation. In Experiment I, the osmolarity decreased at the highest applied concentration by more than 50 units compared to the solvent control. The osmolarity was generally high compared to the physiological level of approximately 300 mOsm. This effect however, is based on a final concentration of 1% DMSO in medium. As the osmolarity is measured by freezing point reduction, 1 % of DMSO has a substantial impact on the determination of osmolarity. The decreased osmolarity does not affect the outcome of the study, because the dose selection was limited by precipitation.
No relevant influence on pH was observed.

CYTOTOXICITY AND CHROMOSOMAL ABERRATIONS:
In Experiment I in the absence and presence of S9 mix, no relevant cytotoxicity was observed up to the highest evaluated concentration, which was limited by test item precipitation.
In Experiment II in the absence of S9 mix after continuous treatment concentrations showing clear cytotoxicity were not evaluable because no evaluable metaphases were present. However, the mitotic index at the highest evaluated concentration was reduced to 51.1 % of control.
Neither with nor without metabolic activation a statistically significant or a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test substance. The aberration rates of the cells after treatment with the test substance (0.0 – 1.7 % aberrant cells, excluding gaps) did not exceed the solvent control values (0.7 – 1.7 % aberrant cells, excluding gaps) and were within the range of the 95 % control limit of the laboratory historical solvent control data. Because the criteria for a clear negative response were met in Experiment I in the presence of S9, a repeat experiment was not necessary. Experiment II, had to be repeated, because the cytotoxicity for the positive controls exceeded the acceptance criteria.

No evidence of an increase in polyploid metaphases was observed after treatment with the test substance as compared to the control cultures.
Either Ethylmethane sulfonate (770.0 or 550.0 µg/mL) or Cyclophosphamide (10.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Table I Summary of Results of the Chromosomal Aberration Study with test substance

Exp	Preparation interval	Test substance concentration (µg/mL)	Mitotic indices (% of control)	Aberrant cells (%) incl. gaps*	Aberrant cells(%) excl.gaps*	Aberrant cells (%)  carrying exchanges
Exposure period 4 h without S9 mix
I	22 h	Solvent control^1	100.0	2.0	1.7	0.0
		Positive control^2	110.6	11.7	11.3^S	1.3
		4.3	96.8	1.0	1.0	0.0
		7.5	105.9	1.0	1.0	0.0
		13.2	120.2	2.3	1.7	0.0
		23.0^P	85.1	0.7	0.7	0.0
Exposure period 22 h without S9 mix
II	22 h	Solvent control^1	100.0	0.7	0.7	0.0
		Positive control^3	52.6	16.3	16.0^S	3.3
		6.4	100.4	1.3	1.0	0.0
		11.2	100.4	0.0	0.0	0.0
		19.6	77.2	1.7	1.7	0.0
		34.4	51.1	0.7	0.7	0.0
Exposure period 4 h with S9 mix
I	22 h	Solvent control^1	100.0	1.3	1.3	0.0
		Positive control^4	82.8	11.0	11.0^S	1.3
		7.5	108.6	1.7	1.7	0.0
		23.0	92.1	1.7	1.7	0.0
		40.3	96.7	1.3	0.7	0.0
		80.6^P	65.6	1.0	1.0	0.0

*  Including cells carrying exchanges

^P  Precipitation occurred at the end oftreatment

^S  Aberration frequency statistically significant higher than corresponding controlvalues

¹ DMSO1.0 % (v/v)

²EMS 770.0µg/mL

³EMS 550.0µg/mL

⁴CPA 10.0µg/mL

Table II Experiment I - Mitotic Index (Preparation Interval 22 h with and without S9 Mix)

Treatment	Conc.	S9	Exposure	Polyploid		Mitotic indices*
group	per mL	mix	period/	cells	Absolute		Mean	%**
			Recovery (h)		1	2
Solv. control^#	1.0 %	-	4 / 18	No polyploidies observed	10.6	8.2	9.4	100.0
Pos. control^##	770.0 µg	-	4 / 18	²	10.9	9.9	10.4	110.6
Test substance	4.3 µg	-	4 / 18	²	10.3	7.9	9.1	96.8
²	7.5 µg	-	4 / 18	²	9.0	10.9	10.0	105.9
²	13.2 µg	-	4 / 18	²	12.2	10.4	11.3	120.2
²	23.0 µg	-	4 / 18	²	6.2	9.8	8.0	85.1
Solv. control^#	1.0 %	+	4 / 18	No polyploidies observed	7.7	7.4	7.6	100.0
Pos. control^###	10.0 µg	+	4 / 18	²	7.0	5.5	6.3	82.8
Test substance	7.5 µg	+	4 / 18	²	9.2	7.2	8.2	108.6
²	23.0 µg	+	4 / 18	²	8.3	5.6	7.0	92.1
²	40.3 µg	+	4 / 18	²	7.4	7.2	7.3	96.7
²	80.6 µg	+	4 / 18	²	5.1	4.8	5.0	65.6

*   The mitotic index was determined in a sample of 1000 cells per culture of each test group in%

^** For the positive control groups and the test substance groups, the relative values of the mitotic index are related to the solvent controls

^#  DMSO

^## EMS

###  CPA

Table III Experiment II - Mitotic Index (Preparation Interval 22 h without S9 Mix)

Treatment	Conc.	S9	Exposure	Polyploid		Mitotic indices*
group	per mL	mix	period/	cells	Absolute		Mean	%**
			Recovery (h)		1	2
Solv. control^#	1.0 %	-	22 / 0	No polyploidies observed	15.0	11.8	13.4	100.0
Pos. control^##	550.0 µg	-	22 / 0	²	7.2	6.9	7.1	52.6
Test substance	6.4 µg	-	22 / 0	²	15.6	11.3	13.5	100.4
²	11.2 µg	-	22 / 0	²	13.8	13.1	13.5	100.4
²	19.6 µg	-	22 / 0	²	12.8	7.9	10.4	77.2
²	34.4 µg	-	22 / 0	²	7.2	6.5	6.9	51.1

*  The mitotic index was determined in a sample of 1000 cells per culture of each test group in%

**  For the positive control groups and the test substance groups, the relative values of the mitotic index are related to the solvent controls

^#   DMSO

^##   EMS

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, test substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating or the highest evaluable concentrations.

Executive summary:

In this GLP study in compliance with OECD 473, the test substance was evaluated using in vitro assay to assess the potential of test substance to induce structural chromosomal aberrations in cultured human lymphocytes in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed.  Per culture 150 metaphases were evaluated for structural chromosomal aberrations. The highest applied concentration in this study (5160.0 µg/mL of the test substance) was chosen with regard to the purity of the test substance meeting and exceeding the maximum requirement by the current OECD Guideline 473 (2014) in order to satisfy EPA guideline OPPTS 870.5375 (1998). Concentration selection for the cytogenetic experiments was performed considering the toxicity data and test substance precipitation and in accordance with OECD Guideline 473 and EC 440/2008 B.10 (2008).  

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which was limited by test substance precipitation.  In Experiment II in the absence of S9 mix after continuous treatment concentrations showing clear cytotoxicity were not evaluable because no evaluable metaphases were present. However, the mitotic index at the highest evaluated concentration was reduced to 51.1 % of control. Neither with nor without metabolic activation was a statistically significant or a biologically relevant increase in the number of cells carrying structural chromosomal aberrations observed after treatment with the test substance. No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance 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 substance did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating or the highest evaluable concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Feb 2019 to 01 Jul 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

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):

CELL LINES
Absence of Mycoplasma contamination: yes
Methods for maintenance in cell culture: suitable for used cell line
Periodically checked for karyotype stability: yes
Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
Type and composition of media, CO2 concentration, humidity level, temperature: RPMI 1640 medium, humidified incubator at 37 ± 1.5 °C in an atmosphere of 4.5% carbon dioxide.

Metabolic activation:

with and without

Metabolic activation system:

S9 PREPARATION
- Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. The protein concentration of the S9 preparation was 30.4 mg/mL

TYPE AND COMPOSITION OF METABOLIC ACTIVATION SYSTEM:
- S9 supernatant was thawed and mixed with S9 co-factor solution to result in a final protein concentration of 0.75 mg/mL in the cultures.
- S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium- ortho-phosphate-buffer (100 mM, pH 7.4).
- S9 mix preparation concentration in the final test medium: 5 % (v/v)

Test concentrations with justification for top dose:

- Experiment I: 7.5, 15.0, 30.0, 45.0, 60.0 µg/mL (-S9/+S9)
- Experiment IC: 12.5, 25.0, 55.0, 60.0 µg/mL (+S9)

Vehicle / solvent:

- Vehicle used: DMSO
- Percentage of solvent in the final culture medium: 0.5 % v/v

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

PRE-TEST FOR CYTOTOXICITY
A pre-test screen was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined by using an osmometer or a pH meter at the maximal concentration of the test substance and in the solvent control without metabolic activation. 1E7 cells in 10 mL RPMI medium are exposed to each concentration of the test substance in the presence and absence of metabolic activation (4 hours). During the 4 hours treatment period the serum concentration is 3%. Following treatment the cells are washed twice by centrifugation (425 g, 10 min) and resuspended in "saline G". Subsequently the cells are resuspended in 30 mL complete culture medium for a 2-day growth period. The cell density is determined each day and adjusted to 3E5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures is 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.

CONCENTRATION SELECTION
Based on the results of the pre-test at least four concentrations were chosen for the mutation experiment. The highest concentration level should be 10 mM or 2 mg/mL as requested by the sponsor, unless limited by the solubility or toxicity of the test substance (relative total growth, RTG). RSG (Relative Suspension Growth, pre-experiment) or RTG (Relative Total Growth) values (main experiment) below 50% are considered toxic. In case of toxic effects, the highest test substance concentration of the main experiment should reduce the parameters of toxicity to approximately 10 - 20%.
To overcome problems with possible deviations in solubility or cytotoxicity, the main experiments were started with more than four concentrations. See Table 1 in 'Any other information on materials and methods incl. tables'.

EXPERIMENTAL PERFORMANCE
In the mutation experiment 1E7 cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3% horse serum were exposed to various concentrations of the test substance either in the presence or absence of metabolic activation. A four hour treatment period was used. After 4 h the test substance was removed by centrifugation (425 × g, 10 min) 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 3E5 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. 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 4E3 cells in selective medium with TFT). The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37 °C ± 1.5°C in 4.5% CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated.

COMPLETE CULTURE MEDIUM
RPMI 1640 medium supplemented with 15% horse serum (3 % HS during 4 hour treatment), 1% of 100 U/100 µg/mL penicillin/ streptomycin, 220 µg/mL sodium-pyruvate, and 0.5 – 0.75% amphotericin used as antifungal.

CLONING MEDIUM
RPMI 1640 (complete culture medium)

SELECTIVE MEDIUM
RPMI 1640 (complete culture medium) by addition of 5 µg/mL TFT.

SALINE G SOLUTION
The "saline G" solution was composed as follows (per litre): 8000 mg NaCl; 400 mg KCl; 1100 mg Glucose; 192 mg Na2HPO4x2H2O; 150 mg KH2PO4; at pH: 7.2

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria:
- The test substance is tested with and without metabolic activation for 4 h.
- An adequate number of cells and at least four concentrations should be analyzable.
- The criteria for the selection of the top concentration are fulfilled.
- The absolute cloning efficiency of the solvent controls at the time of mutant selection (CE) is 65 – 120% (mean value of culture I and II).
- The mean total suspension growth of the solvent control calculated by the day 1 fold- increase in cell number multiplied by the day 2-fold-increase in cell number is 8 – 32.
- The mean mutant frequency of the solvent control is in the range of 50E-6 – 170E-6 cells performed with four parallel cultures.
- The positive controls should yield an absolute increase in mean total MF (Mutation Frequency, number of mutant colonies per 1E6 cells), that is an increase above spontaneous background MF (an induced MF [IMF]) of at least 300E-6 cells. At least 40% of the induced mutation frequency (IMF) should be reflected in the small colony MF. Alternatively, the positive controls should induce at least 150E-6 small colonies.
- The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures (RTG should not be less than 10% of the concurrent selective control group). Furthermore, the mean MF of the positive control must be within or close to the acceptable range established for the laboratory.
- Concentrations which induce a relative adjusted cloning efficiency I below 10% will not be scored for gene mutations.

Statistics:

The statistical analysis was performed with the mean values of culture I and II. A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test substance were 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT
Test substance concentrations between 3.9 µg/mL and 500.0 µg/mL were used in the pre- experiment with and without metabolic activation following 4 hour treatment. The highest concentration was based on the solubility properties of the test substance. Relevant cytotoxic effects were observed at 250.0 µg/mL and above in the absence of metabolic activation and at 62.5 µg/mL and above in the presence of metabolic activation. The test medium was checked for precipitation and phase separation at the end of the treatment period (4 hours) before the test substance was removed. Precipitation occurred at 62.5 µg/mL and above with and without metabolic activation. There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test substance measured in the pre-experiment (solvent control: 354 mOsm, pH 7.29 versus 373 mOsm and pH 7.22 at 500.0 µg/mL). Treatments in the absence of metabolic activation.

MAIN EXPERIMENT !
Based on the results of the pre-experiment the following concentrations were applied in the main experiment without metabolic activation: 0.9; 1.9, 3.8; 7.5; 15.0; 30.0; 45.0; 60.0; 80.0 µg/mL.
Precipitation was noted at 60.0 and 80.0 µg/mL. The cultures at 0.9 – 3.8 µg/mL were not evaluated for mutagenicity as a minimum of only four concentrations is required by the test guideline. The cultures at the highest concentration of 80.0 µg/mL were not continued since the test guideline advises only one precipitating concentration be analysed to avoid artefactual effects. A relative total growth (RTG, calculated as mean of culture I and II) of less than 50 % occurred at precipitating concentration of 60.0 µg/mL. No substantial or reproducible concentration dependent increase in the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed. The statistical analysis was performed based on the mean values of culture I and II. No statistically significant concentration dependent trend was observed. Therefore, the test substance is considered to be non-mutagenic under the conditions of this assay. The mean value of the solvent control was 74 mutant colonies per E6 cells. This value is within the recommended range of 50 – 170 mutant colonies per E6 cells, and therefore considered valid. The range in the groups treated with the test substance was from 69 up to 103 mutant colonies per E6 cells. MMS was used as positive control and showed a distinct increase in induced total mutant colonies demonstrating the correct performance of the assay. A treatment for 24 h in the absence of metabolic activation was not considered to be required due to the nature of the test substance. Treatments in the presence of metabolic activation

MAIN EXPERIMENT 1:
Based on the results of the pre-experiment the following concentrations were applied in the main experiment with metabolic activation: 0.9; 1.9, 3.8; 7.5; 15.0; 30.0; 45.0; 60.0; 80.0 µg/mL.
Precipitation was noted at 80.0 µg/mL. The cultures at 0.9 – 3.8 µg/mL were not evaluated for mutagenicity as a minimum of only four concentrations is required by the guideline. The cultures at the highest concentration of 80.0 µg/mL were not continued due to extreme cytotoxicity. A relative total growth of less than 50 % occurred at 60.0 µg/mL (RTG of 33.0). No substantial or reproducible concentration dependent increase in the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed. No statistically significant concentration dependent trend was observed. The mean value of the solvent control was 87 mutant colonies per E6 cells. This value is within the recommended range of 50 – 170 mutant colonies per E6 cells, and therefore considered valid. The range in the groups treated with the test substance was from 69 up to 99 mutant colonies per E6 cells. CPA was used as positive control and showed a distinct increase in induced total mutant colonies demonstrating the correct performance of the assay.
The precipitating concentration was not included in the evaluation of the experiment due to severe cytotoxic effects. Because at the top concentration evaluated neither the recommended RTG range of 10 – 20 % was reached nor was precipitation present this experimental part was repeated.

MAIN EXPERIMENT 1A:
After the start of the experiment it was realized that instead of two, only one solvent control was used in Experiment 1A, therefore, the experiment was considered invalid and terminated after 4 hours and repeated (Experiment IB) using identical concentrations.

MAIN EXPERIMENT 1B:
The following concentrations were applied in Experiment 1B: 3.13; 6.25; 12.5; 25.0; 50.0; 60.0; 70.0; and 80.0 µg/mL.
Precipitation was noted at 70.0 and 80.0 µg/mL. The cultures at 3.13 µg/mL were not continued for selection as a minimum of only four concentrations is required by the guideline. The cultures at the highest concentration of 80.0 µg/mL were not continued to avoid analysis of too many precipitating concentrations. A relative total growth of less than 50 % occurred at 50.0 µg/mL and above in both cultures. At the next higher concentration of 60.0 µg/mL the RTG was 5.6 in culture I, and 27.4 in culture II. Due to this considerable difference of RTG in both cultures, this experiment was discontinued, and no mutagenicity was analyzed.

MAIN EXPERIMENT 1C:
Based on the toxicity data of Experiment IB, the following concentrations were applied in the repeat Experiment 1C: 3.2; 6.3; 12.5; 25.0; 50.0; 55.0; 60.0; 65.0; 70.0; and 75.0 µg/mL.
Precipitation was noted at 70.0 and 75.0 µg/mL. The cultures at the highest concentration of 75.0 µg/mL were not continued since the cell growth was completely inhibited after 4 hours. The cultures at 3.2 and 6.3 µg/mL were not evaluated for mutagenicity as a minimum of only four concentrations is required by the guideline. The cultures at 70.0 µg/mL could not be evaluated for mutagenicity due to severe cytotoxicity.
No substantial or reproducible concentration dependent increase in the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed. The statistical analysis was performed based on the mean values of culture I and II. No statistically significant concentration dependent trend was observed. The mean value of the solvent control was 54 mutant colonies per E6 cells. This value is within the recommended range of 50 – 170 mutant colonies per E6 cells, and therefore considered valid. The range in the groups treated with the test substance was from 48 up to 68 mutant colonies per E6 cells. The viability of the solvent control of experiment 1C, culture I, with metabolic activation exceeded the upper limit of 120 % (131 %). This deviation was judged as irrelevant as the viability in the parallel culture II was in the acceptable range (113 %).
Viability levels above 100% frequently occur as the cell density of the suspension cell cultures is measured to derive viability. Suspension cell cultures do not necessarily form ideal chemical solutions as individual cells interact with each other forming transient aggregates. The transient aggregates are counted as single cells when measuring cell densities and cause viability levels above 100% as each cell of the aggregates may grow into colonies. CPA was used as positive control and showed a distinct increase in induced total mutant colonies demonstrating the correct performance of the assay.

Conclusions:

Test substance did not demonstrate mutagenic potential in this in vitro cell mutation assay performed in compliance with GLP and following an OECD 490 guideline, and afforded a clearly negative result under the experimental conditions described.

Executive summary:

The study was performed to investigate the potential of test substance to induce mutations at the mouse lymphoma thymidine kinase locus of the cell line L5178Y in compliance with GLP and following an OECD 490 guideline. The assay was performed using the microwell method with and without liver microsomal activation using two parallel cultures with four hours treatment.

Pre-experiment:

Test substance concentrations between 3.9 µg/mL and 500.0 µg/mL were used in the pre- experiment with and without metabolic activation following 4 hour treatment. The highest concentration was based on the solubility properties of the test item. Relevant cytotoxic effects were observed at 250.0 µg/mL and above in the absence of metabolic activation and at 62.5 µg/mL and above in the presence of metabolic activation. The test medium was checked for precipitation and phase separation at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 62.5 µg/mL and above with and without metabolic activation. There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test substance measured in the pre-experiment (solvent control: 354 mOsm, pH 7.29 versus 373 mOsm and pH 7.22 at 500.0 µg/mL).

Treatments in the absence of metabolic activation:

- Main experiment I: Based on the results of the pre-experiment the following concentrations were applied in the main experiment without metabolic activation: 0.9; 1.9, 3.8; 7.5; 15.0; 30.0; 45.0; 60.0; 80.0 µg/mL

Precipitation was noted at 60.0 and 80.0 µg/mL. The cultures at 0.9 – 3.8 µg/mL were not evaluated for mutagenicity as a minimum of only four concentrations is required by the test guideline. The cultures at the highest concentration of 80.0 µg/mL were not continued since the test guideline advises only one precipitating concentration be analysed to avoid artefactual effects. A relative total growth (RTG, calculated as mean of culture I and II) of less than 50 % occurred at precipitating concentration of 60.0 µg/mL. No substantial or reproducible concentration dependent increase in the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed. The statistical analysis was performed based on the mean values of culture I and II. No statistically significant concentration dependent trend was observed. Therefore, test substance tech. is considered to be non-mutagenic under the conditions of this assay. The mean value of the solvent control was 74 mutant colonies per 1E6 cells. This value is within the recommended range of 50 – 170 mutant colonies per 1E6 cells, and therefore considered valid. The range in the groups treated with the test item was from 69 up to 103 mutant colonies per 1E6 cells. MMS was used as positive control and showed a distinct increase in induced total mutant colonies demonstrating the correct performance of the assay. A treatment for 24 h in the absence of metabolic activation was not considered to be required due to the nature of the test substance.

Treatments in the presence of metabolic activation:

- Main experiment 1A:

Based on the results of the pre-experiment the following concentrations were applied in the main experiment with metabolic activation: 0.9; 1.9, 3.8; 7.5; 15.0; 30.0; 45.0; 60.0; 80.0 µg/mL.

Precipitation was noted at 80.0 µg/mL. The cultures at 0.9 – 3.8 µg/mL were not evaluated for mutagenicity as a minimum of only four concentrations is required by the guideline. The cultures at the highest concentration of 80.0 µg/mL were not continued due to extreme cytotoxicity. A relative total growth of less than 50% occurred at 60.0 µg/mL (RTG of 33.0). No substantial or reproducible concentration dependent increase in the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed. No statistically significant concentration dependent trend was observed. The mean value of the solvent control was 87 mutant colonies per 106 cells. This value is within the recommended range of 50 – 170 mutant colonies per 1E6 cells, and therefore considered valid. The range in the groups treated with the test item was from 69 up to 99 mutant colonies per 106 cells. CPA was used as positive control and showed a distinct increase in induced total mutant colonies demonstrating the correct performance of the assay. The precipitating concentration was not included in the evaluation of the experiment due to severe cytotoxic effects. Because at the top concentration evaluated neither the recommended RTG range of 10 - 20% was reached nor was precipitation present this experimental part was repeated.

- Main Experiment 1A:

After the start of the experiment it was realized that instead of two, only one solvent control was used in Experiment IA, therefore, the experiment was considered invalid and terminated after 4 hours and repeated (Experiment 1B) using identical concentrations.

- Main Experiment 1B:

The following concentrations were applied in Experiment 1B: 3.13; 6.25; 12.5; 25.0; 50.0; 60.0; 70.0; and 80.0 µg/mL. Precipitation was noted at 70.0 and 80.0 µg/mL. The cultures at 3.13 µg/mL were not continued for selection as a minimum of only four concentrations is required by the guideline. The cultures at the highest concentration of 80.0 µg/mL were not continued to avoid analysis of too many precipitating concentrations. A relative total growth of less than 50% occurred at 50.0 µg/mL and above in both cultures. At the next higher concentration of 60.0 µg/mL the RTG was 5.6 in culture I, and 27.4 in culture II. Due to this considerable difference of RTG in both cultures, this experiment was discontinued, and no mutagenicity was analyzed.

- Main Experiment 1C:

Based on the toxicity data of Experiment 1B, the following concentrations were applied in the repeat Experiment 1C: 3.2; 6.3; 12.5; 25.0; 50.0; 55.0; 60.0; 65.0; 70.0; and 75.0 µg/mL. Precipitation was noted at 70.0 and 75.0 µg/mL. The cultures at the highest concentration of 75.0 µg/mL were not continued since the cell growth was completely inhibited after 4 hours. The cultures at 3.2 and 6.3 µg/mL were not evaluated for mutagenicity as a minimum of only four concentrations is required by the guideline. The cultures at 70.0 µg/mL could not be evaluated for mutagenicity due to severe cytotoxicity. No substantial or reproducible concentration dependent increase in the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed. The statistical analysis was performed based on the mean values of culture I and II. No statistically significant concentration dependent trend was observed. The mean value of the solvent control was 54 mutant colonies per 106 cells. This value is within the recommended range of 50 – 170 mutant colonies per 1E6 cells, and therefore considered valid. The range in the groups treated with the test substance was from 48 up to 68 mutant colonies per 1E6 cells. CPA was used as positive control and showed a distinct increase in induced total mutant colonies demonstrating the correct performance of the assay.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test substance tech. 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 substance  is considered to be non- mutagenic in this mouse lymphoma assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- in vivo mammalian somatic cell study: cytogenicity/bone marrow chromosome aberration, negative, OECD TG 474, Dunton 2016

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 03 December 2015 (first animal arrival).
Experimental termination date: 04 February 2016 (last day of slide scoring).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

rat

Strain:

other: Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River (UK) Ltd., Margate, Kent, CT9 4LT, England
Age: 6 to 7 weeks at start of experiment
Weight: range 160 to 217 g, mean 181 g
Acclimatisation period: at least 5 days
Housing: up to 3 per cage
Diet: pelleted standard diet, ad libitum
Drinking water: tap water, ad libitum
Temperature: 19 to 24 °C
Humidity: 45 to 64% (81% on one occasion only)
Photoperiod: 12 hours darkness to 12 hours light

Route of administration:

oral: gavage

Vehicle:

0.5% w/v aqueous carboxymethylcellulose with 0.1% v/v Tween 80

Details on exposure:

Vehicle and test substance were administered at a dose volume of 10 mL/kg body weight.
The positive control substance was administered at a dose volume of 5 mL/kg body weight.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Animals received two doses of vehicle alone or test substance in vehicle, approximately 24 hours apart.
Animals treated with the positive control were given a single dose of CPA

Post exposure period:

Animals were observed periodically for 24 hours after the first (all groups) and second dose
(Groups 1 to 4).

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle, negative control

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

CPA, positive control

No. of animals per sex per dose:

Six

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Slide Analysis: A unique, unambiguous code was devised for each main study animal. Adhesive labels that covered the animal and group identity were affixed to each slide so that the analyst could see only the study number and the new code. 2000 polychromatic erythrocytes (PCE), including micronucleated PCE (MN PCE), were counted for each main study animal. The numbers of normochromatic erythrocytes (NCE) and micronucleated NCE (MN-NCE) were also recorded for the first 1000 cells scored. Only areas of slides of good technical quality and appropriate staining characteristics were scored.

Details of tissue and slide preparation:

Slide Preparation: Range-finder animals were killed after the terminal blood sampling, approximately 24 hours after the second administration of the test item. The main study animals in Groups 1 to 4 were killed approximately 24 hours after the second test item or vehicle administration. Group 5 animals were killed approximately 24 hours after the single administration of the positive Control. A single femur was removed from each animal. The bone marrow cells from the femur were aspirated into labelled tubes and centrifuged. The supernatant was withdrawn and the cells were re suspended in a minimal volume of foetal bovine serum. One drop of cell suspension was placed on each of two slides and spread. All slides were left to air dry and age overnight before fixing for five minutes in methanol. Fixed slides were stained for 20 to 30 minutes in 11.5% (v/v) Giemsa in Sorensen’s buffer pH 6.0.

Evaluation criteria:

For the test to have been considered positive i.e. a substance was considered to induce clastogenic/aneugenic damage, the following criteria would have had to be met:
• A statistically significant increase in the frequency of MN-PCE occurred at one or more dose levels.
• The incidence and distribution of MN-PCE in individual animals at the dose level(s) showing statistical significance exceeded the lab's historical negative Control data.
• A dose-related increase in the frequency of MN-PCE (where more than two dose levels are analysed) was observed.
Results which only partially satisfy the above criteria would be dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result.
Biological relevance was taken into account, for example consistency of response within and between dose levels.
A test was considered to be negative, i.e. non-clastogenic/aneugenic, if there was neither a dose-response curve nor any group showed statistically significant increases in the frequency of micronucleated PCEs compared with the negative Controls.

Statistics:

The data were analysed in accordance with the UKEMS guidelines. The data analysed were the proportion of micronucleated PCEs (MN-PCE) and the ratio of polychromatic to normochromatic cells (PCE/NCE).
The proportion of micronucleated PCEs (with respect to the total number of PCEs counted) is considered to be a measure of chromosomal or cell division apparatus damage. The ratio PCE/NCE is considered to provide an estimate of general toxicity of the test item to bone marrow. In this case testing is concerned with a reduction in the PCE/NCE ratio from the vehicle Control value.
The preferred approach for the MN-PCE data was to combine the data within each group and construct a 2x2 contingency table for each treated group with the negative Control. The groups were then compared using a one tailed Fisher Exact test. However, this approach was first validated by carrying out a test for between animal heterogeneity using a chi square test. If the heterogeneity test was significant at the 1% level then an exact Wilcoxon Rank Sum test would be used instead of the Fisher Exact test. The same method was used to compare the positive and negative Controls.
For the PCE/NCE data, the groups given the substance and the positive Control group were compared with the negative Control using one tailed exact Wilcoxon Rank Sum tests.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Definitive test: There were no clinical signs observed following administration of SYN547407 to male rats at dose levels up to 2000 mg/kg/day, nor were there any adverse clinical observations in Group 1 (negative Control) or Group 5 (positive Control) animals.
Exposure to the substance was confirmed in all range-finder blood samples.

Dose-sighting phase: Rapid breathing was observed following the second administration at 500 mg/kg/day, 1250 mg/kg/day and 2000 mg/kg/day. Pale faeces were also observed following administration at 2000 mg/kg/day. Body weight loss was seen in all animals.
Range-finding phase: Rapid breathing was observed following the second administration at 2000 mg/kg/day. One female showed signs of abnormal sensitivity to touch and disturbance followed by slow breathing and piloerection. Body weight loss was seen in all animals.
Based on the results of this phase, the MTD was considered to exceed the limit dose of 2000 mg/kg/day in males and females.

Conclusions:

The substance is considered to be neither clastogenic nor aneugenic in the in vivo rat bone marrow micronucleus assay.

Executive summary:

The substance was tested under GLP to evaluate its potential to cause damage to chromosomes or cell division apparatus, or to cause cell cycle interference, leading to micronucleus formation in polychromatic erythrocytes in the bone marrow of young adult rats following OECD TG 474.
In all phases, the dosing of the vehicle and test item was by oral (gavage) administration twice, approximately 24 hours apart.
In the dose-sighting phase, three groups of two male rats were given the substance as a suspension in 0.5% w/v aqueous carboxymethylcellulose with 0.1% v/v Tween 80 at 500, 1250 or 2000 mg/kg/day, in order to determine the maximum tolerated dose (MTD). In the range-finding phase, a group of three male and three female rats were given the substance at 2000 mg/kg/day, in order to confirm the MTD. The MTD was confirmed to be greater than the limit dose of 2000 mg/kg/day in male and female rats, and as there was no inter-sex difference in toxicity, the main study was conducted in males only.
A proof of exposure phase was conducted to demonstrate that the bone marrow was exposed to the test item, via analysis of test item in the whole blood of treated animals. The presence of substance was confirmed by analysis of the study samples alongside samples of blank matrix and matrix spiked with the test item. Exposure to the test substance was confirmed in all blood samples.
For the main study phase, three groups, each of six male rats were dosed with 500, 1000 or 2000 mg/kg/day test substance. A group of six male rats (negative controls) was dosed with the vehicle alone and a positive control group, also of six male rats, was given a single 15 mg/kg oral (gavage) dose of Cyclophosphamide monohydrate (CPA).
Bone marrow was harvested from all range-finding and main study animals approximately 24 hours after the final dose administration and smears were prepared. The stained slides prepared for the main study were coded and 2000 polychromatic erythrocytes (PCE) per animal were scored for the presence of micronuclei and the group frequencies were statistically analysed.
There were no statistically significant increases in micronucleus frequency in male rats treated at any dose level of of the substance, compared with the negative control group. There was no evidence of a statistically significant reduction in the PCE/NCE ratio in male rats treated with the substance and, since proof of exposure to the bone marrow was demonstrated in the range finding phase of the study, this indicated a lack of toxicity of the substance to the bone marrow.
The animals dosed with CPA, the positive control item, had statistically significant increases in the number of micronucleated cells compared with the concurrent vehicle control group, which demonstrated that the test system was capable of detecting a known clastogen and that the scorers were capable of detecting micronuclei. There was a statistically significant decrease in the PCE/NCE ratio in the positive control group, indicating toxicity to the bone marrow.
In conclusion, it can be stated that there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the substance up to the OECD 474 limit dose of 2000 mg/kg/day in male rats. The substance is considered to be neither clastogenic nor aneugenic in the rat bone marrow micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro OECD TG 471:

A reliable and valid Ames test was performed under GLP to OECD TG 471 to investigate the potential of test substance to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II and IIa) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA (pKM101) and WP2 (pKM101). The plates incubated with the test item showed normal background growth up to the maximal dose of 5000 µg/plate with and without S9 mix in all strains used. Minor cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in Experiment I in strain WP2 uvrA (pKM101) at 5000 µg/plate with and without S9 mix. No further cytotoxic effects were observed, neither in the remaining test groups of Experiment I, nor in Experiments II and IIa. No relevant increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no observed 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, which showed a distinct increase of induced revertant colonies.

A second study was carried out under GLP to OECD TG 471 to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains. Only in experiment II a minor reduction in the number of revertants was observed in strain WP2 uvrA pKM101 in the presence of S9 mix at 5000 µg/plate. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations and all mutation rates were within the range of normal biological variability. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In vitro OECD TG 473:

The test substance was evaluated under GLP to OECD TG 473 using an vitro assay to assess the potential to induce structural chromosomal aberrations in cultured human lymphocytes in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed and 150 metaphases per culture were evaluated for structural chromosomal aberrations. The highest applied concentration in this study (5160.0 µg/mL of the test substance) was chosen with regard to the purity of the test substance to satisfy EPA guideline OPPTS 870.5375 (1998). Concentration selection for the cytogenetic experiments was performed considering the toxicity data and test substance precipitation.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which was limited by test substance precipitation. In Experiment II in the absence of S9 mix after continuous treatment concentrations showing clear cytotoxicity were not evaluable because no evaluable metaphases were present. However, the mitotic index at the highest evaluated concentration was reduced to 51.1% of control. Neither with nor without metabolic activation was a statistically significant or a biologically relevant increase in the number of cells carrying structural chromosomal aberrations observed after treatment with the test substance. No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance 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 vitro OECD TG 490:

This reliable and valid study was performed under GLP to OECD TG 490 to investigate the potential of test substance to induce mutations at the mouse lymphoma thymidine kinase locus of the cell line L5178Y. The microwell method with and without liver microsomal activation was applied using two parallel cultures with four hours treatment.
A pre-experiment with a treatment period of 4 hours was performed with and without metabolic activation to determine cytotoxic effects and precipitation of test substance. Relevant cytotoxic effects were observed at 250.0 µg/mL and above in the absence of metabolic activation and at 62.5 µg/mL and above in the presence of metabolic activation. Precipitation occurred at 62.5 µg/mL and above with and without metabolic activation. There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test substance measured in the pre-experiment (solvent control: 354 mOsm, pH 7.29 versus 373 mOsm and pH 7.22 at 500.0 µg/mL). Treatments in the absence of metabolic activation in the main experiment without metabolic activation were performed at 0.9, 1.9, 3.8, 7.5, 15.0, 30.0, 45.0, 60.0 and 80.0 µg/mL. Precipitation was noted at 60.0 and 80.0 µg/mL. No substantial or reproducible concentration dependent increase in the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed. No statistically significant concentration dependent trend was observed. Therefore, the substance was considered to be non-mutagenic under the conditions of this assay. The mean value of mutant colonies in the solvent control of 74 per 1E6 cells was within the recommended range of 50 – 170 mutant colonies, and therefore considered valid. The range in the groups treated with the test item was from 69 up to 103 mutant colonies per 1E6 cells. MMS was used as positive control and showed a distinct increase in induced total mutant colonies demonstrating the correct performance of the assay. A treatment for 24 h in the absence of metabolic activation was not considered to be required due to the nature of the test substance.

Treatments in the presence of metabolic activation in the main experiment with metabolic activation were carried out at 0.9, 1.9, 3.8, 7.5, 15.0, 30.0, 45.0, 60.0 and 80.0 µg/mL. Precipitation was noted at 80.0 µg/mL. The cultures at the highest concentration of 80.0 µg/mL were not continued due to extreme cytotoxicity. A relative total growth of less than 50% occurred at 60.0 µg/mL (RTG of 33.0). No substantial or reproducible concentration dependent increase in the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed. No statistically significant concentration dependent trend was observed. The mean value of the solvent control was 87 mutant colonies per 106 cells. This value is within the recommended range of 50 – 170 mutant colonies per 1E6 cells, and therefore considered valid. The range in the groups treated with the test item was from 69 up to 99 mutant colonies per 106 cells. CPA was used as positive control and showed a distinct increase in induced total mutant colonies demonstrating the correct performance of the assay. The precipitating concentration was not included in the evaluation of the experiment due to severe cytotoxic effects. At the top concentration evaluated, the recommended RTG range of 10 - 20% was not reached and no precipitation was present. This experimental part was repeated with adopted concentrations until a valid experiment was achieved. No substantial or reproducible concentration dependent increase in the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed. The statistical analysis was performed based on the mean values of culture I and II. No statistically significant concentration dependent trend was observed. The mean value of the solvent control was 54 mutant colonies per 106 cells and considered valid as within the recommended range of 50 – 170 mutant colonies per 1E6 cells. The range in the treatment groups was from 48 up to 68 mutant colonies per 1E6 cells. CPA was used as positive control and showed a distinct increase in induced total mutant colonies demonstrating the correct performance of the assay.

A second reliable GLP study performed to OECD TG 490 confirmed that the substance was not inducing mutations at the thymidine kinase locus of the cell line L5178Y when tested with and without S9 metabolic activation.

In vivo OECD TG 474: 

The substance was tested under GLP to evaluate its potential to cause damage to chromosomes or cell division apparatus, or to cause cell cycle interference, leading to micronucleus formation in polychromatic erythrocytes in the bone marrow of young adult rats following OECD TG 474.
A proof of exposure phase was conducted to demonstrate that the bone marrow was exposed to the test item, via analysis of test item in the whole blood of treated animals. The presence of substance was confirmed by analysis of the study samples alongside samples of blank matrix and matrix spiked with the test item. Exposure to the test substance was confirmed in all blood samples.
In the main study, three groups of six male rats each were dosed twice by oral gavage), approximately 24 hours apart with 500, 1000 or 2000 mg/kg/day test substance. A group of six male rats (negative controls) was dosed with the vehicle (0.5% w/v aqueous carboxymethylcellulose with 0.1% v/v Tween 80) alone and a positive control group, also of six male rats, was given a single 15 mg/kg oral (gavage) dose of Cyclophosphamide monohydrate (CPA).
Bone marrow was harvested from all range-finding and main study animals approximately 24 hours after the final dose administration and smears were prepared. The stained slides prepared for the main study were coded and 2000 polychromatic erythrocytes (PCE) per animal were scored for the presence of micronuclei and the group frequencies were statistically analysed.
There were no statistically significant increases in micronucleus frequency in male rats treated at any dose level of of the substance, compared with the negative control group. There was no evidence of a statistically significant reduction in the PCE/NCE ratio in male rats treated with the substance and, since proof of exposure to the bone marrow was demonstrated in the range finding phase of the study, this indicated a lack of toxicity of the substance to the bone marrow.
The animals dosed with CPA, the positive control item, had statistically significant increases in the number of micronucleated cells compared with the concurrent vehicle control group, which demonstrated that the test system was capable of detecting a known clastogen and that the scorers were capable of detecting micronuclei. There was a statistically significant decrease in the PCE/NCE ratio in the positive control group, indicating toxicity to the bone marrow.

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 (EC) No. 1272/2008.