Pirimiphos-methyl

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, according to OECD TG 471, Sokolowski 2015

- In vitro chromosome aberration test, +S9 negative, -S9 negative, Human lymphocytes, according to OECD TG 473, Sokolowski 2015

- In vitro gene mutation assay, +S9 negative, -S9 negative, Chinese hamster V79 cells, according to OECD TG 476, Naumann 2017

- In vitro sister chromatid exchange, +S9 positive, -S9 positive, Chinese hamster lung fibroblast, equivalent to EPA-560/6 -84 -002, Richardson 1986

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:

14 Jan 2015 to 30 Jan 2015

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), 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/ β-naphthoflavone induced rat liver S9
- Method of preparation of S9 mix: The S9 was prepared from 8 – 12 weeks old male Wistar rats (RjHan:WI; weight approx. 220 – 320 g, induced by peroral administration of 80 mg/kg b.w. phenobarbital and by peroral administrations of β-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.
- Concentration or volume of S9 mix and S9 in the final culture medium: S9 supernatant was 10 % v/v in the S9 mix. S9 mix contains: 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: Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo[a]pyrene. The protein concentration in the S9 preparation was 39.3 mg/mL in both experiments.

Test concentrations with justification for top dose:

Experiment I (-S9 and +S9): 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II (-S9 and +S9): 10; 33; 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

other: 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 PERFOMANCE
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 100 μL test solution (solvent or 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 72 hours at 37 °C in the dark, plates were then stored at 4 °C until counted.
* 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 the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and 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). Due to precipitation of the test item the colonies were partly counted 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 a significant increase in mutant colony frequencies
- 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 exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration 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 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.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

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

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 pKM101, WP2 uvrA 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 and time of the determination: Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in experiment I and in experiment II from 2500 to 5000 µg/plate in all strains with S9 mix and at 5000 µg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording.

STUDY RESULTS
- Signs of toxicity: Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (μg/plate) shown in table 1 in 'Any other information on results incl. tables'
- Mean number of revertant colonies per plate and standard deviation: No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration, 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.

Table 1. Toxic effects, evident as a reduction in the number of revertants

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	5000	/	/	/
TA 98	2500 – 5000	5000	/	/
TA 100	/	/	/	/
WP2 pKM101	/	5000	/	/
WP2uvrApKM101	/	/	/	/

/= no toxic effects

Conclusions:

The test substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This AMES test was performed under GLP following the OECD 471 guideline 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.

Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in experiment I and in experiment II from 2500 to 5000 µg/plate in all strains with S9 mix and at 5000 µg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test substance showed normal background growth in all strains used up to the highest concentration. Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I in strain TA 1537 without S9 mix, strain TA 98 with and without S9 mix and strain WP2 pKM101with S9 mix. 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 conclusion, it can be stated that during the described mutagenicity test 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 this 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:

17 Dec 2014 to 17 Mar 2015

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:

2014

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 blood

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Blood samples were obtained from healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (30 years old) for Experiment IA, from a female donor (38 years old) for Experiment IB and from a female donor (26 years old)
for Experiment II. Blood samples were drawn by venous puncture and collected in heparinized tubes.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hour 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 and 1.10 mL whole blood. All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Colcemid (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/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system.
- 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. 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).
- Quality controls of S9: 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 27.6 mg/mL

Test concentrations with justification for top dose:

Absence of S9 mix
Experiment 1A: 31.3, 62.5, 125.0 µg/ mL
Experiment 1B: 15.6, 31.3, 62.5, 150.0 µg/ mL
Experiment 2: 31.3, 62.5, 125.0 µg/ mL

Presence of S9 mix
Experiment 1A: 62.5, 125.0, 250.0, 500.0 µg/mL
Experiment 2: 31.3, 125.0, 250.0, 375.0 µg/mL

Vehicle / solvent:

- Solvent used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent: The solvent was chosen due to its solubilisation properties and its relative non-toxicity to the cell cultures.

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:

Culture condition: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs 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.

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".
- 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 metaphases: 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 using 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 150 well-spread metaphases per culture (total 300) were scored for cytogenetic damage on coded slides, except for the positive controls in Experiment II with S9 mix, where only 75 metaphases were evaluated. Only metaphases with 46 ± 1 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 protocol. The results were presented in tabular form, including experimental groups with the test substance, solvent controls, and positive controls, respectively.

Evaluation criteria:

A test substance is classified as non-clastogenic if:
a. none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. there is no concentration-related increase when evaluated with an appropriate trend test,
c. all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)
A test substance is classified as clastogenic if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) The increase is dose-related when evaluated with an appropriate trend test,
c) Any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits)

Statistics:

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

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:

In the absence and presence of S9 mix, no clastogenicity was observed at the concentrations evaluated. None of the aberration rates of the cells after treatment with the test substance in Experiment IA with and without S9 mix and Experiment IB and II without S9 mix (0.0 – 2.7 % aberrant cells, excluding gaps) were statistically significant or above the range of the laboratory historical solvent control data. In Experiment II in the presence of S9 mix, small increases in chromosomal aberrations were observed that exceeded the two-fold standard deviation 95 % control limit (2.6 % aberrant cells, excluding gaps) of the laboratory historical solvent control data (3.5, 2.7 and 3.8% at 31.3, 125.0 and 250.0 µg/ml respectively). Since these values were not statistically significant with respect to the concurrent solvent control and no dose-dependency was observed, these findings are considered to be biologically irrelevant. Furthermore, these effects were not observed in Experiment IA under exactly the same conditions, even at higher concentrations inducing a higher level of cytotoxicity.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures.
Either EMS (770.0 or 825.0 µg/mL) or CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations in agreement with the laboratory control data.
Overview of the results were provided in table 1 and table 2 in “Any other information on results incl. tables”

Table 1. Summary of the results of the chromosomal aberration study with the test substance (exposure period 4 h)

Exp	Preparation interval (h)	Test item concentration (µg/mL)	Mitotic indices (% of control)	incl. gaps*	Aberrant cells* excl. gaps* (%)	carrying exchanges
Exposure period 4 h without S9 mix
IA	22	Solvent control^1	100.0	1.7	1.3	0.0
		Positive control^2	80.1	8.3	8.3^S	2.7
		31.3^T	95.4	1.3	1.0	0.0
		62.5^T	80.7	3.0	2.3	0.0
		125.0^T	59.2	1.0	1.0	0.0
IB	22	Solvent control^1	100.0	1.0	0.7	0.0
		Positive control^3#	49.0	8.5	8.2^S	1.3
		15.6	75.3	1.7	1.3	0.0
		31.3	68.2	3.3	2.3	0.0
		62.5^T/PS	56.9	2.3	2.0	0.0
		150^T/PS	43.9	2.3	1.7	0.0

Table 2. Summary of the results of the chromosomal aberration study with the test substance (exposure period 22 h)

Exp	Preparation interval (h)	Test item concentration (µg/mL)	Mitotic indices (% of control)	incl. gaps*	Aberrant cells* excl. gaps* (%)	carrying exchanges
Exposure period 22 h without S9 mix
II	22	Solvent control^1	100.0	2.0	1.3	0.0
		Positive control^2	59.5	25.3	25.3^S	8.3
		31.3	97.7	1.7	1.7	0.0
		62.5^#	58.8	2.0	2.0	0.0
		125.0^PS	47.9	0.3	0.3	0.0

^* Including cells carrying exchanges

^# Evaluation of 300 metaphases per culture

^## Evaluation of 75 metaphases per culture

^PS Phase separation occurred at the end of treatment / T Turbidity

^S Aberration frequency statistically significant higher than corresponding control values

¹ DMSO 1.0 % (v/v)

² EMS 770.0 μg/mL

³ EMS 825.0 μg/mL

Table 3. Results osmolarity and pH in exp IA, IB and II

		Concentration [µg/mL]	Osmolarity [mOsm]	pH-value
Exp. IA	Solvent control	-	470	7.7
	Test substance	2000.0	452	7.7
Exp. IB	Solvent control	-	473	7.6
	Test substance	250.0	466	7.6
Exp. II	Solvent control	-	470	7.5
	Test substance	250.0	469	7.4

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, the test substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic concentrations.

Executive summary:

This chromosome aberration study performed under GLP and following OECD TG 473 assessed the potential of the 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/β-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed. Per culture at least 150 metaphases were evaluated for structural chromosomal aberrations, except for the positive controls in Experiment II with S9 mix, where only 75 metaphases were evaluated. The highest applied concentration in this study (2000.0 μg/mL of the test substance) was chosen. Concentration selection for the cytogenetic experiments was performed considering the toxicity data and test substance turbidity.

In the absence and presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentrations. In the absence and presence of S9 mix, no clastogenicity was observed at the concentrations evaluated. In Experiment II in the presence of S9 mix, small increases in chromosomal aberrations were observed that exceeded the two-fold standard deviation 95 % control limit (2.6 % aberrant cells, excluding gaps) of the laboratory historical solvent control data (3.5, 2.7 and 3.8 % at 31.3, 125.0 and 250.0 μg/mL respectively). Since these values were not statistically significant with respect to the concurrent solvent control and no dose-dependency was observed, these findings are considered to be biologically irrelevant. Furthermore, these effects were not observed in Experiment IA under exactly the same conditions, even at higher concentrations inducing a higher level of cytotoxicity. 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, at concentrations exhibiting acceptable cytotoxicity as measured by the mitotic index in agreement with the laboratory control data. Furthermore, no relevant influence on the osmolarity or pH was observed.  The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is likely 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.  This was deemed to have no implication on study conduct.

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 cytotoxic concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 Feb 2017 to 19 Jun 2017

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 using the Hprt and xprt genes)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a sufficiently high cloning efficiency of untreated cells (as a rule more than 50%) are both necessary properties for the appropriate performance of the study, and both attributes are present in this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Cell culture conditions:
Thawed stock cultures are propagated at 37 °C in 75 cm^2 plastic flasks. About 2-3×10^6 cells are seeded into each flask with 15 mL of minimal essential medium (MEM) containing Hank’s salts supplemented with 10% fetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells are sub-cultured once or twice weekly. The cell cultures are incubated at 37 °C in a 1.5% carbon dioxide atmosphere (98.5% air).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/β-naphthoflavone induced rat liver S9
- method of preparation of S9 mix : The S9 was prepared from 8 – 12 weeks old male Wistar rats induced by peroral administration of 80 mg/kg bw 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
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentration of the S9 preparation in the experimental parts with metabolic activation was 30.1 mg/mL
- quality controls of S9: 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.

Test concentrations with justification for top dose:

In absence of metabolic activity:
- Experiment I: 35.0; 46.7; 58.4; 70.0; and 105.0 µg/mL.
- Experiment II: 53.8; 70.0; 85.0; 105.0; and 125.0 µg/mL

In presence of metabolic activity:
Experiment I: 17.5; 35.0, 44.3; 53.0; and 61.8 µg/mL.
Experiment II + III: 25.0; 45.0; 50.0; 55.0; and 60.0 µg/mL

Vehicle / solvent:

- Solvent used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent: The solvent was chosen according to its solubilisation properties and its compatibility with cell cultures
- Justification for percentage of solvent in the final culture medium: The final concentration of DMSO in the culture medium was 0.5% (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Pre-test for cytotoxicity
A pre-test was performed in order to determine the toxicity of the test item to the cell line. In addition the pH-value and the osmolarity was measured. In the pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is evident as a reduction of the cloning efficiency (CE).

Seeding
Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in phosphate buffered saline (PBS). Prior to the trypsin treatment the cells were treated with PBS containing 200 mg/L EDTA. Approximately 0.7 to 1.2×10^7 cells were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment. With the cell doubling time of approximately 12 h this ensures a population of more than 20×10^6 cells treated with the test substance.

Treatment
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G''. The pH was adjusted to 7.2. Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×10^6 cells per experimental point (concentration series plus controls) were sub-cultured in 175 cm² flasks containing 30 mL medium. Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2. The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below. Three days after first sub-cultivation approximately 2.0x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of approximately 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5x10^5 cells each in medium containing 6-TG (11 μg/mL). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2. After 7 – 10 days the colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Colonies with more than 50 cells were counted. If in doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent solvent control in both parallel cultures,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical solvent control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing) could be useful.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results. Therefore the test chemical response should be concluded to be equivocal (interpreted as equally likely to be positive or negative).

Statistics:

The statistical analysis was performed in the mean values of culture I and II. A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependency of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A t-test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval and other if necessary. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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 item concentrations between 17.2 μg/mL and 2200 μg/mL were used in the pre-experiment. The highest concentration was chosen with respect to the current OECD Guideline 476 (2016) and the purity of the test item.
Relevant cytotoxic effects were observed at 68.8 μg/mL and above in the presence and absence of metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Phase separation occurred at 137.5 μg/mL and above with and without metabolic activation. There was no relevant shift of the osmolarity and pH value even at the maximum concentration of the test substance in the pre-experiment

Treatments in the absence of metabolic activation
Experiment I: The concentration range of Experiment I was based on the data of the pre-experiment. Experiment I was evaluated at the following concentrations: 35.0; 46.7; 58.4; 70.0; and 105.0 μg/mL. Phase separation was noted at 105.0 μg/mL. Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I below 50 % (mean value of both parallel cultures) occurred at 105.0 μg/mL. No increase in mutant frequency (MF) outside the 95 % control limits of the solvent historical control data was observed. The linear regression analysis was negative. However, as the optimal cytotoxicity cut-off range was not achieved, an additional experiment was conducted using an adjusted concentration range.
Experiment II: Experiment II was evaluated at the following concentrations: 53.8; 70.0; 85.0; 105.0; and 125.0 μg/mL. Phase separation was noted at 105.0 μg/mL and above. Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (RS) below 50 % (mean value of both parallel cultures) occurred at 125.0 μg/mL and above. In the absence of metabolic activation no increase in MF outside the 95 % control limits of the solvent historical control data was observed. The linear regression analysis was negative. Therefore the criteria for a clearly negative response were met in the absence of metabolic activation.

Treatments in the presence of metabolic activation
Experiment I: The concentration range of Experiment I was based on the data of the pre-experiment. Experiment I was evaluated at the following concentrations: 17.5; 35.0, 44.3; 53.0; and 61.8 μg/mL. Phase separation was noted at 61.8 μg/mL with S9 mix. Strong cytotoxic effects indicated by a relative adjusted cloning efficiency below 10 % occurred at 61.8 μg/mL accompanied by phase separation. Hence, the recommended cytotoxic range of approximately 10-20 % relative adjusted cloning efficiency 1 (RS) was not achieved. The 95 % control limit of the historical control data MF was marginally exceeded at concentrations of 44.3 and 53.0 μg/mL, although the increases in MF were within the overall HCD range (3.4 – 41.0). A t-test performed at these concentrations showed statistically significant increases in MF. A linear regression analysis, performed on the concentrations showing acceptable cytotoxicity, was positive. Since the recommended cytotoxic range of approximately 10-20 % RS was not achieved in Experiment I, a second experiment was performed with adjusted concentrations.
Experiment II: Experiment II was evaluated at the following concentrations: 25.0; 45.0; 50.0; 55.0; and 60.0 μg/mL. Phase separation was noted at 60.0 μg/mL and above. Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (RS) below 50 % (mean value of both parallel cultures) occurred at 45.0 μg/mL and above. Strong cytotoxic effects indicated by a relative adjusted cloning efficiency below 10 % occurred at the two highest evaluated concentrations of 55.0 μg/mL and 60.0 μg/mL. Since only three concentrations that fulfilled the acceptance criteria with respect to cytotoxicity were available in Experiment II, this experimental part was repeated (Experiment III) using adjusted concentrations.
Experiment III: Experiment III was evaluated at the following concentrations: 25.0; 45.0; 50.0; 55.0; and 60.0 μg/mL. Relevant cytotoxic effects occurred at 50.0 μg/mL and above. The recommended cytotoxic range of approximately 10-20% relative adjusted cloning efficiency I (RS) was achieved. Increases in MF outside the 95 % control limit of the historical control data were observed at 25.0, 45.0; 55.0, and 60.0 μg/mL. A t-test showed a statistically significant increase in MF at 60.0 μg/mL. The linear regression analysis however was negative indicating no concentration related effect. Overall, the increases in mutation frequency were sporadic and not concentration related nor reproducible in parallel cultures, therefore can be considered to be of limited biological significance and not indicative of mutagenicity. In the presence of metabolic activation, the test substance is considered not to give a mutagenic response under the conditions of this assay. EMS and DMBA were used as positive control agents and showed a distinct increase in induced mutant colonies (MF).

Conclusions:

In conclusion, under the experimental conditions the test item did not induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in the presence and absence of metabolic activation. Therefore, the test substance is not considered to be mutagenic in this HPRT assay.

Executive summary:

The test substance was assessed (according OECD TG 476 and GLP principles) for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The assay was performed in three independent experiments, using two parallel cultures each. The treatment time was 4 hours. Experiment I and II were performed with and without metabolic activation. Experiment III was performed solely with metabolic activation. Treatments in absence of metabolic activation were evaluated at the following concentrations: 35.0; 46.7; 58.4; 70.0; and 105.0 µg/mL (experiment I) and 53.8; 70.0; 85.0; 105.0; and 125.0 µg/mL (experiment II)

Results of experiment I showed that phase separation was noted at 105.0 µg/mL. Relevant cytotoxic effects occurred at 105.0 µg/mL. The optimal cytotoxicity cut-off range was not achieved, an additional experiment was conducted using an adjusted concentration range. For experiment II, phase separation was noted at 105.0 µg/mL and above. Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (RS) below 50 % (mean value of both parallel cultures) occurred at 125.0 µg/mL and above. In the absence of metabolic activation no increase in MF outside the 95 % control limits of the solvent historical control data was observed. The linear regression analysis was negative. Therefore the criteria for a clearly negative response were met in the absence of metabolic activation

Treatments in presence of metabolic activation were evaluated at the following concentrations: 17.5; 35.0, 44.3; 53.0; and 61.8 µg/mL (experiment I) and 25.0; 45.0; 50.0; 55.0; and 60.0 µg/mL (experiment II and III),

Results of experiment I showed that a strong cytotoxic effect (relative adjusted cloning efficiency below 10 %) and phase separation occurred at 61.8 µg/mL. Hence, the recommended cytotoxic range of approximately 10-20 % relative adjusted cloning efficiency 1 (RS) was not achieved. Since the recommended cytotoxic range of approximately 10-20 % RS was not achieved, a second experiment was performed with adjusted concentrations. For experiment II,

relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (RS) below 50 % (mean value of both parallel cultures) occurred at 45.0 µg/mL and above. Strong cytotoxic effects indicated by a relative adjusted cloning efficiency below 10 % occurred at the two highest evaluated concentrations of 55.0 µg/mL and 60.0 µg/mL. Since only three concentrations fulfilled the acceptance criteria in Experiment II, this experimental part was repeated (Experiment III) using adjusted concentrations. For experiment III, relevant cytotoxic effects occurred at 50.0 µg/mL and above. The recommended cytotoxic range of approximately 10-20 % relative adjusted cloning efficiency I (RS) was achieved. Increases in MF outside the 95 % control limit of the historical control data were observed at 25.0, 45.0; 55.0, and 60.0 µg/mL. A t-test showed a statistically significant increase in MF at 60.0 µg/mL. The linear regression analysis however was negative indicating no concentration related effect. Overall, the increases in mutation frequency were sporadic and not concentration related nor reproducible in parallel cultures, therefore can be considered to be of limited biological significance and not indicative of mutagenicity. In the presence of metabolic activation, the test substance is considered not to give a mutagenic response under the conditions of this assay. EMS and DMBA were used as positive control agents and showed a distinct increase in induced mutant colonies (MF).

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in the presence and absence of metabolic activation. Therefore, the test substance is not considered to be mutagenic in this HPRT assay.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

7 Oct 1985 to 30 Oct 1985

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Remarks:

The OECD test guideline for this type of test was deleted because of a lack of understanding of the mechanism(s) of action of the effect detected by the test. It is therefore considered to be an unsuitable test system to investigate genetic toxicity.

Qualifier:

equivalent or similar to guideline

Guideline:

other: Test Guidelines. EPA-560/6-84-002.

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Don cells (lung, diploid; Chinese hamster, Cricetulus griseus). The karyotype was checked shortly before the start of experimentation with the majority of cells having 22 chromosomes, abnormal (marker ) chromosomes were not observed.

MEDIA USED
- Type and composition of media: The cells were cultured in sodium bicarbonate buffered Dulbecco's MEM (D-MEM) containing 10 % foetal calf serum (FCS) and 10 units/mL penicillin/streptomycin solution and 2 mM glutarnine. The cells were maintained at 37 °C ± 1.5 °C throughout culture using a flow CO2 incubator.

Cytokinesis block (if used):

10 µg/mL Colchicine

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Ten male Sprague-Dawley rats were each given an intraperitoneal injection of AROCLOR 1254, 500 mg/kg from a working solution of 200 mg/mL in corn oil.
- Method of preparation of S9 mix: The livers were washed with several changes of ice-cold buffer, by decanting, until the washings were free from blood. The pieces were then transferred using sterile forceps to a weighed beaker containing 100 mL of buffer. The beaker was re-weighed and the liver weight
noted. A 25% w/v liver homogenate was to be made and so the volume of buffer to be used was calculated as three times the liver weight. Weight of liver = 70 mg, volume buffer 3 x 70 = 210 mL. The homogeniser tube was rinsed out with sterile buffer and liver pieces added to it together with buffer from the measuring cylinder. The liver was homogenised with 8 passes (one stroke down, one stroke up= 1 pass) of the pestle rotating at 1,070 rpm. The homogenate was poured into a sterile 500 mL conical, stood on ice. The procedure was repeated, with swabbing of the pestle with 70 % meths, until all the liver had been homogenised. The homogenate was made up to the correct volume with the rest of the buffer and mixed by swirling the contents. Equal volumes (40 mL maximum) were added by means of a sterile pipette to pairs of centrifuge tubes which were kept on ice. Each tube was capped tightly, and then placed in a pre-cooled 8 x 50 mL rotor. The rotor was centrifuged at 9000 x gin an MSE 21 centrifuge with the temperature set at 4 °C for 10 minutes. The timing sequence was started once the rotor had reached full speed. When the centrifuge had stopped, the tubes were removed and placed on ice. The supernatant (S-9) was carefully decanted off into a conical flask.

Test concentrations with justification for top dose:

0.14 - 289 μg/mL of the test substance

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.5-1.5 x 10^6 cells
- Test substance added in medium: Cells were treated with the test substance during exponential growth when the monolayer was approximately 60% confluent. The test substance was dissolved in DMSO at room temperature, and dosed in a volume of 30 μL to duplicate cultures at 8 dose levels to give a concentration range of 0.14 - 289 μg/mL culture medium.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours

PROCESSING OF CULTURES:
a) 300µL of a solution of colchicine in physiological saline was added to cultures approximately 2 hours prior to harvest to give a final concentration of 10 µg/mL.
b) In the 24th hour after culture treatment the growth medium from each flask was removed and discarded. Residual serum was washed from the monolayer using 10ml physiological saline. This washing was collected in appropriately labelled UNIVERSAL containers. Cells were dissociated from the monolayer with 5ml of 0.25% trypsin in EDTA, this process took between 2-5 minutes. Following dissociation, 5ml of D-MEM containing 10% FCS was added to neutralise enzyme activity, the resulting cell suspension was added to the saline washings collected previously and centrifuged at 300g for 5 minutes.
c) The Don cells were re-suspended in 5ml of 0.075M KCl and timing of 7 minutes commenced. The cell suspensions during this time were transferred to labelled 15ml conical centrifuge tubes and a further 5cm^3 0.075M KCl was added. This process was carried out at room temperature and was followed by centrifugation at 300g for 5 minutes.
d) The cells were then fixed in acetic acid/methanol (1 part glacial acetic acid ; 3 parts methanol): 1ml was added dropwise to the pellet which was agitated using a WHIRLIMIX, and a further 9ml of fixative was then added. After at least one hour the fixative was changed 3 times to remove any residual crystals of KCl.
e) Several drops of a 'cloudy’ concentration of fixative and cells (in most cases this was approximately 1 mL) were dropped from about 10-30 cm using a Pasteur pipette, onto clean dry microscope slides, and air-dried. At least four slides were made from each culture, with extra slides prepared to test staining procedures, i.e. to determine optimum Giemsa concentration and timings.

STAINING TECHNIQUE:
a) A single slide from each culture was stained in 10% Giemsa in distilled water and the mitotic index determined.
b) Prior to staining, the prepared slides from selected dose levels were stored at room temperature in darkness for at least one week.
c) The chromosome preparations were stained in a 10 µg/mL solution of Hoescht fluorochrome (bisbenzamid 33258) in 2% KCl for 15 minutes at room temperature, and rinsed thoroughly in distilled water.
d) Coverslips were mounted in water onto the slides and sealed in place around the edges with COPYDEX rubber solution.
e) The stained microscope slides were then exposed to a UV lamp at approximately a 30cm distance for approximately 1.5 hours.
f) Coverslips were removed, and slides stained in 3% Giemsa stain (in distilled water) for 5 minutes. Slides were then mounted in DPX.
g) Appropriate dose levels were selected for SCE analysis, the highest dose level being either one indicative of the toxic limit (i.e. reduced mitosis), or showing increased chromosomal aberrations, plus at least 2 lower doses. From each selected culture, 25-50 cells were analysed for the incidence of SCL The slides were coded to avoid observer bias during analysis. The exception being a positive control culture where analysis of 10 cells was sufficient to confirm a positive SCE-inducing response. Data was recorded at each exchanged point, and not as the area between exchanges, i.e. a terminal exchange was counted as a single SCE, all others as two.
h) The ratio of M1 : M2 : M3 cells (first, second and third mitosis cells) was calculated to evaluate any effects the chemical had on cell cycle kinetics particularly on cells in second division.

CRITERIA FOR DETERMINING A POSITIVE RESULT:
In determining a positive result the following criteria were satisfied:
i) Statistically significant difference from solvent controls at the p<0.01 level, in at least one dose level.
ii) Evidence of a dose-response relationship.
iii) The relationship between statistical and biological significance will have been considered before a final evaluation is made.

Statistics:

Results were expressed as units of mean SCE per cell and mean SCE per chromosome. Statistical analysis was performed on data relating to SCE/cell. It was considered unnecessary to perform the same analysis on data relating SCE/chromosome. A Student's one-sided t-test was used to compare mean SCE/cell from the test substance treated cultures (and positive controls) with solvent control values.

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

positive

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:

Results for cultures without metabolic activation:
The ratios of first:second:third metaphase cells, (M 1:M2:M3), were determined for all cultures. If second division cells are reduced by 20% or more in treated cultures this is considered to be indicative of the test compound being
biologically active in the system, and an acceptable criterion for selection as an appropriate maximum tolerated dose level. At 289 and 145 µg/mL there were no cells available for scoring in second divisions and at 29 and 14 µg/mL the number of cells in second metaphase was reduced by greater than 20% when compared to solvent control values. The appropriate cultures selected for SCE analysis were 29, 14, 2.9, 1.4, 0.29 and 0.14 µg/mL of the test substance.
For interpretation and statistical analyses, data from duplicate cultures at each treatment level were combined. Substantially elevated and statistically significant (p<0.01) increases in SCE/cell were observed in cultures treated with 29, 14, 1.4 and 0.14 µg/mL of the test substance. A statistically significant difference from solvent control values was not observed at the 2.9 µg/mL dose level, with statistical significance reduced (p<0.05) at the 0.29 µg/mL level.
The test system was shown to be sensitive to agents which induce SCE in the absence of auxiliary metabolic activation as demonstrated by the response obtained by mitomycin C.

Results for cultures with metabolic activation:
The ratios of M1:M2:M3 cells were determined for the solvent and positive control cultures and for the cultures treated with the test substance. At the 289 µg/mL dose level there were no cells available in second division. At the 145 µg/ml level in a single culture there were no cells available in second division, although, in the other 145 µg/ml culture sufficient cells in second metaphase were available and analysed for SCE. This dose level was not considered appropriate as a MTD because of the high level of cytotoxicity. The dose selected as the appropriate MTD for the test substance was 29 µg/mL where second division cells were depressed by approximately 20%. The appropriate cultures selected for SCE analysis were 29, 14, 2.9, 1.4, 0.29 and 0.14 µg/mL of the test substance. The single culture of 145 µg/mL with second division cells was also analysed to provide additional information as to compound-effects outside of the upper margin of cytotoxicity.
The results of the SCE analyses are expressed in both units of SCE/cell and SCE/chromosome. For interpretation and statistical analyses, data from duplicate cultures at each treatment level were combined relating to SCE/cell. Statistical analysis was again confined to data Substantially elevated and statistically significant (p<0.01) increases in SCE/cell were observed in the upper
part of the dose range, (145) 29 and 14 µg/mL. There were no statistically significant increases in SCE/cell in cultures treated with 2.9, 1.4, 0.29 or 0.14 µg/mL of the test substance.

Positive control:
The test system was shown to be sensitive to agents which induce SCE as a consequence of auxiliary metabolic activation as demonstrated by the response obtained by cyclophosphamide.

Overview of the results were provided in table 1 and table 2 in “Any other information on results incl. tables”

Table 1. Results expressed as mean SCE/cell and mean SCE/chromosome from control cultures and cultures treated with the test substance without S-9 mix.

Treatment	SCE/Cell	SCE/Chromosome
DMSO Control: 1 µl/mL	7.65	0.34
Mitomycin C: 0.1 µg/mL	25.42**	1.12
Test substance
29 µg/mL	13.34**	0.61
14 µg/mL	11.38**	0.51
2.9 mg/mL	8.40	0.37
1.4 µg/mL	13.32**	0.58
0.29 µg/mL	9.76*	0.41
0.14 µg/mL	10.64**	0.49

Table 2. Results expressed as mean SCE/cell and mean SCE/chromosome from control cultures and cultures treated with the test substance with S-9 mix.

Treatment	SCE/Cell	SCE/Chromosome
DMSO Control: 1 µL/mL	7.96	0.37
Cyclophosphamide: 5.0 µg/mL	96.66**	4.20
Test substance
145 µg/mL	19.32**	0.83
29 µg/mL	10.31**	0.47
14 µg/mL	9.96**	0.45
2.9 µg/mL	7.24	0.34
1.4 µg/mL	6.84	0.31
0.29 µg/mL	8.42	0.38
0.14 µg/mL	8.12	0.39

Conclusions:

It is concluded the test substance induces SCE in Chinese hamster cells in vitro both in presence and absence of auxiliary metabolic activation, although, a dose response relationship is not firmly established.

Executive summary:

To assess the sister chromatid exchange (SCE) inducing potential of the test substance was assessed in a study, performed under GLP following the EPA-560/6-84-002 guideline. Chinese hamster lung fibroblasts (Don cells) were exposed in vitro to 0.14, 0.29, 1.4, 2.9, 14, 29, 145 and 189 µg/mL, both in the absence and presence of auxiliary metabolic activation (S-9 mix). The maximum dose tested in each case was determined by the cytotoxicity of the compound, a dose of 29 µg/mL being selected as the maximum dose level for both phases of the study.

The test system was shown to be sensitive to SCE-inducing effects by the response given to the positive control substances, 0.1 µg/mL mitomycin C (direct acting SCE-inducer) and 5.0 µg/mL cyclophosphamide (an SCE-inducer requiring metabolic activation). In the absence of any auxiliary metabolic activation, statistically significant increases in SCE/cell were seen in cultures treated with 29, 14, 1.4, 0.29 and 0.14 µg/mL. A statistically significant increase was not seen in cultures treated with 2.9 μg/mL. Thus no clear dose response relationship was evident. In the presence of auxiliary metabolic activation, statistically significant increases in SCE frequency were observed at the two higher dose levels, 29 and 14 µg/mL (p<0.01). There were no statistically significant differences from SCE control values in any cultures treated with lower dose levels of the test substance. A single culture of 145 μg/mL which exceeded the appropriate MTD was analysed, and at a high level of cytotoxicity SCE formation was increased more than 2-fold.

It is concluded the test substance induces SCE in Chinese hamster cells in vitro both in presence and absence of auxiliary metabolic activation, although, a dose response relationship is not firmly established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus test in mouse, negative, according to OECD 474, Flanders 2017

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:

1 Mar 2017 to 16 Jun 2017

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:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: Hsd:ICR (CD-1)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Six to ten weeks old
- Weight at study initiation: 22.1 to 30.9 g
- Housing: The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 1 Mar 2017 to 16 Jun 2017

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Carboxymethyl-cellulose (CMC)
- Justification for choice of vehicle: 0.5% CMC was used as the vehicle as it gave an emulsion suitable for dosing.
- Amount of vehicle: 10 mL/kg
- Concentration of test material in vehicle: 17.5, 35 and 70 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was freshly prepared as required as an emulsion at the appropriate concentration in 0.5% CMC.

Frequency of treatment:

Single dose

Post exposure period:

24 hours and 48 hours (highest dose group only)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test.
- Route of administration: Single oral administration
- Actual dose: 50 mg/kg
- Dose volume: 10 mL/kg

Tissues and cell types examined:

Bone marrow, Polychromatic erythrocytes (PCEs)

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

SLIDE EVALUATION: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

METHOD OF ANALYSIS: A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the corresponding vehicle control group.

CRITERIA FOR DOSE SELECTION:
A range-finding toxicity test was performed to determine a suitable dose level for the main test. The dose level selected should ideally be the maximum tolerated dose level or a maximum recommended dose of 2000 mg/kg if no clinical signs are observed. The range- finding toxicity test was also used to determine if the main test was to be performed using both sexes or males only. Bone marrow samples were taken from the range-finding animals 48 hours after dosing and slides were prepared and qualitatively assessed to ensure that any bone marrow toxicity observed was within acceptable limits for the main test. In addition, three satellite groups of three male mice were dosed with the maximum proposed dose level for the main test alongside the confirmatory range-finding dose level. These animals were terminated at 1, 4, and 24 hours after dosing and blood samples were taken to provide plasma and blood cell samples for proof of exposure analysis. The main study would only be performed if the Study Director and Sponsor agreed that the analysis from the satellite groups indicated that exposure to the bone marrow had been achieved.

Evaluation criteria:

Acceptability criteria
The test is considered acceptable if the following criteria are met:
- The concurrent negative control data are considered acceptable for addition to the laboratory historical control database
- The concurrent positive controls or scoring controls should induce responses that are compatible with those generated in the historical control database and produce a statistically significant increase compared with the concurrent negative control
-The appropriate number of doses and cells has been analysed

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range-Finding Toxicity Test
In animals dosed with the test item at 2000 mg/kg, premature death was observed in the male animal and the following clinical signs were observed: hunched posture, ptosis, tiptoe gait, and emaciation. At 1000 mg/kg, the following clinical signs were observed: hunched posture, ptosis, lethargy, ataxia, splayed gait, tonic convulsions, and emaciation. The male animal was euthanized due to the severity of the clinical signs. Clinical signs of hunched posture and ptosis were observed in animals dosed at 300 and 600 mg/kg. In animals dosed at 800 mg/kg the following clinical signs were observed: hunched posture, ptosis, tonic convulsions, splayed gait, and emaciation. In animals dosed at 700 mg/kg the clinical signs included hunched posture, ptosis and splayed gait. Emaciation was also observed in one animal at this dose level. However, following discussions with the animal technician, the emaciation was considered to be due to the other animals not allowing this animal to feed. Qualitative analysis of the bone marrow slides indicated that there was no evidence of any significant toxicity to the bone marrow in any of the range-finding animals. The micronucleus test was therefore conducted, following agreement from the Sponsor and the Study Director, using the oral route in groups of seven mice (males) at the considered maximum tolerated dose of 700 mg/kg, with 350 and 175 mg/kg as the two lower dose levels. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice.

Proof of exposure
Analysis of the plasma and blood cell samples indicated that the test item was present in the satellite group animals at all timepoints and that bone marrow exposure would most likely be achieved.

Micronucleus test
There were no premature deaths observed in any of the dose groups in the main test. Clinical signs of hunched posture and ptosis were observed in the 350 mg/kg dose group. In the 700 mg/kg dose groups, the clinical signs were more severe than those observed at the same dose in the range-finding test and included: hunched posture, ptosis, lethargy, ataxia, splayed gait, hypothermia, elevated tail, decreased respiratory rate, laboured respiration, increased salivation, occasional body tremors, and increased respiratory rate. However, the more severe clinical signs were only observed at the 24 or 48-hour observations and were only observed in 2 of the animals in the 24-hour dose groups, and 2 of the animals in the 48-hour dose group. The reason for this was not known however it may be considered to be due to variable sensitivity of the animals to the test item.

Evaluation of bone marrow slides
There were no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group.
There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item dose groups when compared to the vehicle control group and were well within the negative control historical control data. Since none of the values for micronucleus frequency in pirimiphos-methyl treated groups were statistically significant, and all were within the HCD, a trend test was not conducted.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

See Table 1 in ‘Any other information on results incl. tables'.

Table 1. Micronucleus test -summary of group mean data

Treatment Group	Number of PCE with Micronuclei per 4000 PCE		PCE/NCE Ratio
	Group Mean	SD	Group Mean	SD
Vehicle Control (0.5% CMC) 10 mL/kg 24-hour Sampling Time	1.6	2.1	0.56	0.28
Positive Control (Cyclophosphamide) 50 mg/kg 24-hour Sampling Time	85.8***	42.0	0.84	0.14
Test substance 700 mg/kg 48-hour Sampling Time	2.4 (P = 0.262)	1.1	0.41 (P=0.295)	0.16
Test substance 700 mg/kg 24-hour Sampling Time	3.4 (P = 0.206)	2.5	0.73	0.46
Test substance 350 mg/kg 24-hour Sampling Time	2.3 (P = 0.464)	1.6	0.67	0.32
Test substance 175 mg/kg 24-hour Sampling Time	2.3 (P = 0.390)	1.3	0.66	0.21

Conclusions:

The test substance was considered to be non-genotoxic under the conditions of the test.

Executive summary:

The micronucleus test was conducted under GLP following the OECD 474 guideline, using the oral route in groups of 7 mice (males) at the maximum tolerated dose of 700 mg/kg, with 350 and 175 mg/kg as the two lower dose levels using 0.5% carboxymethyl-cellulose (0.5% CMC) as the vehicle. A range-finding test was performed to find suitable dose levels of the test item, and to investigate if there was a marked difference in toxic response between the sexes. In addition, three satellite groups of three male mice were dosed with the maximum proposed dose level for the main test alongside the confirmatory range-finding dose level. These animals were terminated at 1, 4, and 24 hours after dosing and blood samples were taken to provide plasma and blood cell samples for proof of exposure analysis. The analysis samples indicated that the test item was present in the plasma and blood cells of the satellite group animals and that bone marrow exposure would most likely be achieved. Validation of the analytical method was performed by spiking known amounts of the test item into control plasma and blood cells and determining accuracy and precision data to meet SANCO/3029/99 guideline. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. Animals were killed after 24 hours and 48 hours (highest dose level only), the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Additional groups of 5 mice were given a single oral dose of 0.5% CMC or dosed orally with cyclophosphamide (50 mg/kg bw), to serve as vehicle and positive controls respectively. Vehicle and positive control animals were terminated after 24 hours.

There were no premature deaths observed in any of the dose groups in the main test. The clinical signs of hunched posture and ptosis were observed in the 350 mg/kg dose group. The clinical signs observed in the 700 mg/kg dose groups were more severe than those observed at 700 mg/kg in the range-finding test and the following were observed: hunched posture, ptosis, lethargy, ataxia, splayed gait, hypothermia, elevated tail, decreased respiratory rate, laboured respiration, increased salivation, occasional body tremors, loss of righting reflex, and increased respiratory rate. However, the more severe clinical signs were only observed at the 24 or 48-hour observations and were only observed in 2 of the animals in the 24-hour dose group, and 2 of the animals in the 48-hour dose group. The reason for this was not known however it may be considered to be due to variable sensitivity of the animals to the test item. There were no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group. There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

In conclusion, the test substance was considered to be non-genotoxic under the conditions of the test. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro OECD 471 (Sokolowski 2015)
This AMES test was performed under GLP following the OECD 471 guideline 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.
Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in experiment I and in experiment II from 2500 to 5000 µg/plate in all strains with S9 mix and at 5000 µg/plate in all strains without S9 mix. The undissolved particles had no influence on the data recording. The plates incubated with the test substance showed normal background growth in all strains used up to the highest concentration. Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I in strain TA 1537 without S9 mix, strain TA 98 with and without S9 mix and strain WP2 pKM101with S9 mix. 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 conclusion, it can be stated that during the described mutagenicity test 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 this Salmonella typhimurium and Escherichia coli reverse mutation assay.

In vitro OECD 473 (Sokolowski 2015)
This chromosome aberration study performed under GLP and following OECD TG 473 assessed the potential of the 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/β-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed. Per culture at least 150 metaphases were evaluated for structural chromosomal aberrations, except for the positive controls in Experiment II with S9 mix, where only 75 metaphases were evaluated. The highest applied concentration in this study (2000.0 μg/mL of the test substance) was chosen. Concentration selection for the cytogenetic experiments was performed considering the toxicity data and test substance turbidity.
In the absence and presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentrations. In the absence and presence of S9 mix, no clastogenicity was observed at the concentrations evaluated. In Experiment II in the presence of S9 mix, small increases in chromosomal aberrations were observed that exceeded the two-fold standard deviation 95 % control limit (2.6 % aberrant cells, excluding gaps) of the laboratory historical solvent control data (3.5, 2.7 and 3.8 % at 31.3, 125.0 and 250.0 μg/mL respectively). Since these values were not statistically significant with respect to the concurrent solvent control and no dose-dependency was observed, these findings are considered to be biologically irrelevant. Furthermore, these effects were not observed in Experiment IA under exactly the same conditions, even at higher concentrations inducing a higher level of cytotoxicity. 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, at concentrations exhibiting acceptable cytotoxicity as measured by the mitotic index in agreement with the laboratory control data. Furthermore, no relevant influence on the osmolarity or pH was observed.  The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is likely 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. This was deemed to have no implication on study conduct.
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 cytotoxic concentrations.

In vitro OECD 476 (Naumann 2017)
The test substance was assessed (according OECD TG 476 and GLP principles) for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The assay was performed in three independent experiments, using two parallel cultures each. The treatment time was 4 hours. Experiment I and II were performed with and without metabolic activation. Experiment III was performed solely with metabolic activation. Treatments in absence of metabolic activation were evaluated at the following concentrations: 35.0; 46.7; 58.4; 70.0; and 105.0 µg/mL (experiment I) and 53.8; 70.0; 85.0; 105.0; and 125.0 µg/mL (experiment II)
Results of experiment I showed that phase separation was noted at 105.0 µg/mL. Relevant cytotoxic effects occurred at 105.0 µg/mL. The optimal cytotoxicity cut-off range was not achieved, an additional experiment was conducted using an adjusted concentration range. For experiment II, phase separation was noted at 105.0 µg/mL and above. Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (RS) below 50 % (mean value of both parallel cultures) occurred at 125.0 µg/mL and above. In the absence of metabolic activation no increase in MF outside the 95 % control limits of the solvent historical control data was observed. The linear regression analysis was negative. Therefore the criteria for a clearly negative response were met in the absence of metabolic activation
Treatments in presence of metabolic activation were evaluated at the following concentrations: 17.5; 35.0, 44.3; 53.0; and 61.8 µg/mL (experiment I) and 25.0; 45.0; 50.0; 55.0; and 60.0 µg/mL (experiment II and III),
Results of experiment I showed that a strong cytotoxic effect (relative adjusted cloning efficiency below 10 %) and phase separation occurred at 61.8 µg/mL. Hence, the recommended cytotoxic range of approximately 10-20 % relative adjusted cloning efficiency 1 (RS) was not achieved. Since the recommended cytotoxic range of approximately 10-20 % RS was not achieved, a second experiment was performed with adjusted concentrations. For experiment II, relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (RS) below 50 % (mean value of both parallel cultures) occurred at 45.0 µg/mL and above. Strong cytotoxic effects indicated by a relative adjusted cloning efficiency below 10 % occurred at the two highest evaluated concentrations of 55.0 µg/mL and 60.0 µg/mL. Since only three concentrations fulfilled the acceptance criteria in Experiment II, this experimental part was repeated (Experiment III) using adjusted concentrations. For experiment III, relevant cytotoxic effects occurred at 50.0 µg/mL and above. The recommended cytotoxic range of approximately 10-20 % relative adjusted cloning efficiency I (RS) was achieved. Increases in MF outside the 95 % control limit of the historical control data were observed at 25.0, 45.0; 55.0, and 60.0 µg/mL. A t-test showed a statistically significant increase in MF at 60.0 µg/mL. The linear regression analysis however was negative indicating no concentration related effect. Overall, the increases in mutation frequency were sporadic and not concentration related nor reproducible in parallel cultures, therefore can be considered to be of limited biological significance and not indicative of mutagenicity. In the presence of metabolic activation, the test substance is considered not to give a mutagenic response under the conditions of this assay. EMS and DMBA were used as positive control agents and showed a distinct increase in induced mutant colonies (MF).
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in the presence and absence of metabolic activation. Therefore, the test substance is not considered to be mutagenic in this HPRT assay.

EPA-560/6-84-002 (Richardson 1986) - disregarded due to unsuitable test system
To assess the sister chromatid exchange (SCE) inducing potential of the test substance was assessed in a study, performed under GLP following the EPA-560/6-84-002 guideline. Chinese hamster lung fibroblasts (Don cells) were exposed in vitro to 0.14, 0.29, 1.4, 2.9, 14, 29, 145 and 189 µg/mL, both in the absence and presence of auxiliary metabolic activation (S-9 mix). The maximum dose tested in each case was determined by the cytotoxicity of the compound, a dose of 29 µg/mL being selected as the maximum dose level for both phases of the study.
The test system was shown to be sensitive to SCE-inducing effects by the response given to the positive control substances, 0.1 µg/mL mitomycin C (direct acting SCE-inducer) and 5.0 µg/mL cyclophosphamide (an SCE-inducer requiring metabolic activation). In the absence of any auxiliary metabolic activation, statistically significant increases in SCE/cell were seen in cultures treated with 29, 14, 1.4, 0.29 and 0.14 µg/mL. A statistically significant increase was not seen in cultures treated with 2.9 μg/mL. Thus no clear dose response relationship was evident. In the presence of auxiliary metabolic activation, statistically significant increases in SCE frequency were observed at the two higher dose levels, 29 and 14 µg/mL (p<0.01). There were no statistically significant differences from SCE control values in any cultures treated with lower dose levels of the test substance. A single culture of 145 μg/mL which exceeded the appropriate MTD was analysed, and at a high level of cytotoxicity SCE formation was increased more than 2-fold.
It is concluded the test substance induces SCE in Chinese hamster cells in vitro both in presence and absence of auxiliary metabolic activation, although, a dose response relationship is not firmly established.

The OECD test guideline for this type of test was deleted because of a lack of understanding of the mechanism(s) of action of the effect detected by the test. It is therefore considered to be an unsuitable test system to investigate genetic toxicity.

In vivo OECD TG 474 (Flanders 2017)
The micronucleus test was conducted under GLP following the OECD 474 guideline, using the oral route in groups of 7 mice (males) at the maximum tolerated dose of 700 mg/kg, with 350 and 175 mg/kg as the two lower dose levels using 0.5% carboxymethyl-cellulose (0.5% CMC) as the vehicle. A range-finding test was performed to find suitable dose levels of the test item, and to investigate if there was a marked difference in toxic response between the sexes. In addition, three satellite groups of three male mice were dosed with the maximum proposed dose level for the main test alongside the confirmatory range-finding dose level. These animals were terminated at 1, 4, and 24 hours after dosing and blood samples were taken to provide plasma and blood cell samples for proof of exposure analysis. The analysis samples indicated that the test item was present in the plasma and blood cells of the satellite group animals and that bone marrow exposure would most likely be achieved. Validation of the analytical method was performed by spiking known amounts of the test item into control plasma and blood cells and determining accuracy and precision data to meet SANCO/3029/99 guideline. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. Animals were killed after 24 hours and 48 hours (highest dose level only), the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Additional groups of 5 mice were given a single oral dose of 0.5% CMC or dosed orally with cyclophosphamide (50 mg/kg bw), to serve as vehicle and positive controls respectively. Vehicle and positive control animals were terminated after 24 hours.
There were no premature deaths observed in any of the dose groups in the main test. The clinical signs of hunched posture and ptosis were observed in the 350 mg/kg dose group. The clinical signs observed in the 700 mg/kg dose groups were more severe than those observed at 700 mg/kg in the range-finding test and the following were observed: hunched posture, ptosis, lethargy, ataxia, splayed gait, hypothermia, elevated tail, decreased respiratory rate, laboured respiration, increased salivation, occasional body tremors, loss of righting reflex, and increased respiratory rate. However, the more severe clinical signs were only observed at the 24 or 48-hour observations and were only observed in 2 of the animals in the 24-hour dose group, and 2 of the animals in the 48-hour dose group. The reason for this was not known however it may be considered to be due to variable sensitivity of the animals to the test item. There were no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group. There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.
In conclusion, the test substance was considered to be non-genotoxic under the conditions of the test. 

 

Justification for classification or non-classification

A small increase in sister chromatid exchange was observed in the in vitro chromatid exchange (SCE) assay in Chinese hamster lung fibroblasts (Richardson 1986). This increase was not considered to be toxicologically significant, since they occurred only at excessively toxic concentrations. It should be noted that the OECD test guideline for the SCE assay has been deleted because the biological origin and relevance of SCE formation for genotoxicity is not fully understood. The use of this assay is no longer promoted by the OECD. Consequently, the SCE assay is not used for regulatory purposes and this study is not considered relevant for the assessment of the genotoxic potential of the test substance.
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.