Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is considered to not induce genetic mutations in bacteria nor in mammalian cells as confirmed in two GLP compliant in vitro studies according to OECD 471 and 476. The test item is also not considered to cause chromosomal aberrations as indicated by results of an OECD 473 and GLP compliant study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-02-08 to 2016-04-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Lot. No.: 10H016
- Suitability of cells: The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes them suitable for gene toxicity assays with low background aberrations.
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 h
- Modal number of chromosomes: 2n = 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 °C in a humidified atmosphere containing 5 % CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated fetal bovine serum (final concentration 10 %).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Concentrations were chosen based on a preliminary cytotoxicty test:
Experiment A
150, 125, 100, 50 µg/mL
Experiment B
75, 50, 25, 12.5, 1.5 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: This vehicle is compatible with the survival of the V79cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10^5 cells/dish

DURATION
- Exposure duration: 3 h with metabolic activation; 20 h without metabolic acitvation
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells): 1.5 cell cycles (20 h, without S9 mix only) and at approximately 2 normal cell cycles (28 h, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

SPINDLE INHIBITOR: colchicine (0.2 μg/mL) 2.5-3 hours prior to harvest.

STAIN: 5 % Giemsa

NUMBER OF REPLICATIONS: 2 plates/concesntration

NUMBER OF CELLS EVALUATED: 300

DETERMINATION OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations were listed with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) were recorded.
– Individual culture data were summarised in tabular form.

Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative because:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.

Statistics:

For statistical analysis, Fisher exact and CHI2 tests were utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no differences to negative control
- Effects of osmolality: no differences to negative control
- Precipitation: There was no precipitation in the medium at any concentration tested.

RANGE-FINDING/SCREENING STUDIES: In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection (cytotoxicity assay) was performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see table 1. - 5. in "any other information on results"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

Table 1. 3 h/20 h treatment/sampling time without S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Ethyl methanesulfonate)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	4.59	2.16	39.84	32.16
SD	1.84	1.37	5.01	4.02
Lower confidence interval	0.00	0.00	23.89	19.38
Upper confidence interval	10.44	6.52	55.80	44.94
n	4	4	4	4

 

Table 2. 3 h/20 h treatment/sampling time with S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Cyclophosphamide)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	5.44	2.34	50.34	44.53
SD	1.73	0.62	2.53	4.35
Lower confidence interval	0.00	0.38	42.31	30.71
Upper confidence interval	10.95	4.31	58.38	58.36
n	4	4	4	4

 

Table 3. 20 h/20 h treatment/sampling time without S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Ethyl methanesulfonate)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	4.94	2.34	45.28	39.75
SD	1.06	0.79	4.11	2.55
Lower confidence interval	1.55	0.00	32.21	31.64
Upper confidence interval	8.32	4.87	58.35	47.86
n	4	4	4	4

 

 

Table 4. 20 h/28 h treatment/sampling time without S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Ethyl methanesulphonate)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	5.06	2.16	45.94	40.69
SD	0.87	0.36	4.61	12.43
Lower confidence interval	2.31	1.01	31.27	28.26
Upper confidence interval	7.82	3.03	60.61	53.11
n	4	4	4	4

 

Table 5. 3 h/28 h treatment/sampling time with S9-mix

	number of aberrant cells/150 cells
	Negative control		Positive control (Cyclophosphamide)
	Incl. Gaps	Excl. Gaps	Incl. Gaps	Excl. Gaps
Mean	4.97	2.16	47.91	40.59
SD	0.36	0.94	2.81	3.10
Lower confidence interval	3.82	0.00	38.97	30.72
Upper confidence interval	6.12	5.14	56.84.	50.47
n	4	4	4	4

 

Conclusions:

The test item tested up to cytotoxic concentrations, both with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.
Therefore, the test item is considered as not clastogenic in this system.

Executive summary:

The test item, was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 50, 100, 125 and 1501 μg/mL

with S9 mix: 100, 150, 175 and 200 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 12.5, 25, 50 and 751 μg/mL

Experiment B with 20/28 h treatment/sampling time without S9 mix: 12.5, 25, 50 and 75 1 μg/mL

Experiment B with 3/28 h treatment/sampling time with S9 mix: 100, 150, 175 and 200 μg/mL

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test was shown as the concurrent positive controls Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) caused the expected increases in cells with structural chromosome aberrations and were compatible with the historical control range.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-03-04 to 2016-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use, June 2012

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– to trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Initial Test:
-S9 Mix: 3000; 1600; 500; 160; 50 and 16 μg/plate;
+S9 Mix: 5000; 1600; 500; 160; 50 and 16 μg/plate;

Confirmatory Test:
Salmonella typhimurium TA98 and TA100 strains:
-S9 Mix: 1000; 500; 160; 50; 16; and 5 μg/plate;
+S9 Mix: 3000; 1600; 500; 160; 50 and 16 μg/plate;
Salmonella typhimurium TA1535 strain:
-S9 Mix: 1250; 750; 500; 160; 50; 16 and 5 µg/plate;
+S9 Mix: 1600; 500; 160; 50; 16 and 5 μg/plate;
Salmonella typhimurium TA1537 strain:
-S9 Mix: 1000; 500; 160; 50; 16 and 5 μg/plate;
+S9 Mix: 1600; 500; 160; 50; 16 and 5 μg/plate.
The examined concentration levels were not be changed in the case of Escherichia coli WP2 uvrA:
-S9 Mix: 3000; 1600; 500; 160; 50 and 16 μg/plate;
+S9 Mix: 5000; 1600, 500; 160; 50 and 16 μg/plate.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: DMSO is a common vehicle recommended in respective guidelines. The test item was completely dissolvable in DMSO up to a concentration of 100 µg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine

Remarks:

without S9; vehicle: DMSO; strain: Salmonella TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; vehicle: ultrapure water; strain: Salmonella TA100, 1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9; vehicle: DMSO; strain: Salmonella TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; vehicle: ultrapure water; strain: E. coli WP2 uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9; vehicle: DMSO; strains:all of Salmonella strains and E.coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony counts; affected background lawn

Rationale for test conditions:

According to guidelines

Evaluation criteria:

The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 750 µg/plate and above (-S9); at 1600 µg/plate and above (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1600 (+S9) and at 500 µg/plate (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate and above (-S9); at 3000 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 160 µg/plate and above (-S9); at 1600 µg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 mg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

RANGE-FINDING/SCREENING STUDIES: In an Initial Mutation Test the selection of the concentrations was done on the basis of a Solubility Test and a concentration range finding test (Informatory Toxicity Test). In the Initial Mutation Test all of the obtained higher revertant colony numbers (higher than the revertant colony numbers of the vehicle control) remained within the corresponding historical control data ranges. In the Initial Mutation Test inhibitory effect of the test item was observed. The cytotoxicity was indicated by decreased revertant colony counts and/or affected background lawn development.

HISTORICAL CONTROL DATA
please see "Any other information on results" table 1

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity was indicated by decreased revertant colony counts and/or affected background lawn development.

Table 1 Historical control values for revertants/plate (for the period of 2008-2015)

		Bacterial strains
Historical control data of DMSO control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	20.9	101.4	10.3	7.9	24.9
		SD	3.5	26.2	1.4	2.5	4.9
		Minimum	10	65	3	2	11
		Maximum	39	150	23	20	44
	+S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	27.1	114.7	12.0	8.8	34.2
		SD	4.0	19.3	1.5	2.1	5.2
		Minimum	15	71	4	3	16
		Maximum	48	161	24	20	56
Historical control data of ultrapure water control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	22.4	105.5	10.4	7.5	26.3
		SD	3.6	27.6	1.6	2.3	5.9
		Minimum	12	67	3	2	13
		Maximum	36	156	24	16	47
	+S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	28.0	117.4	11.5	8.7	35.2
		SD	4.0	19.8	1.4	2.3	5.2
		Minimum	15	83	4	4	18
		Maximum	43	166	22	16	56
Historical control data of positive control	-S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	255.6	958.9	842.1	467.4	712.3
		SD	30.7	149.9	134.0	105.7	57.5
		Minimum	123	522	354.0	109	320
		Maximum	647	1927	1871	1498	1283
	+S9		TA 98	TA 100	TA 1535	TA 1537	E. coli
		Average	1224.8	1431.9	165.4	148.0	264.7
		SD	293.8	339.9	35.1	21.3	74.2
		Minimum	409	581	85	68	141
		Maximum	2587	2923	507	407	487

 

Table 2 Summary Table of the Results of the Range Finding Test

Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimurium tester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	19.0	1.14	33.0	1.39	85.7	1.00	109.3	1.00
DMSO Control (50 µL)	16.7	1.00	23.7	1.00	85.7	1.00	99.0	1.00
DMSO Control (100 µL)	19.3	1.00	21.3	1.00	–	–	104.0	1.00
Ultrapure Water Control	–	–	–	–	76.3	1.00	–	–
5000	0.3	0.02	9.0	0.38	1.7	0.02	18.3	0.19
1600	10.7	0.64	24.0	1.01	67.0	0.78	92.0	0.93
500	20.0	1.20	20.3	0.86	90.7	1.06	99.7	1.01
160	15.3	0.92	26.7	1.13	98.0	1.14	112.0	1.13
50	15.3	0.92	25.0	1.06	96.3	1.12	101.0	1.02
16	17.7	1.06	25.3	1.07	91.3	1.07	99.0	1.00
5	14.3	0.86	29.0	1.23	97.0	1.13	102.7	1.04
NPD (4mg)	269.7	13.95	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1337.3	17.52	–	–
2AA (2mg)	–	–	1170.7	54.88	–	–	1392.0	13.38

Table 3 Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	23.0	1.11	24.0	1.14	95.0	1.03	102.7	1.08	10.0	1.07	9.7	1.45	11.0	1.18	10.7	1.14	22.3	1.10	22.3	1.05
DMSO Control	20.7	1.00	21.0	1.00	92.7	1.00	95.0	1.00	9.3	1.00	6.7	1.00	9.3	1.00	9.3	1.00	20.3	1.00	21.3	1.00
Ultrapure Water Control	–	–	–	–	83.3	1.00	–	–	8.3	1.00	–	–	–	–	–	–	17.0	1.00	–	–
5000	–	–	6.7^SB	0.32	–	–	2.7^SB	0.03	–	–	0.7^SB	0.10	–	–	0.0^B	0.00	–	–	15.0^SB	0.70
3000	2.3^B	0.11	–	–	2.0^B	0.02	–	–	0.0^B	0.00	–	–	0.0	0.00^B	–	–	16.3^SB	0.80	–	–
1600	3.3^B	0.16	18.7	0.89	27.7^SB	0.30	75.7	0.80	0.0^B	0.00	4.7	0.70	0.7	0.07^B	3.7	0.39	19.7	0.97	22.0	1.03
500	20.3	0.98	26.0	1.24	99.3	1.07	99.3	1.05	9.3	1.00	8.7	1.30	2.7	0.29	10.0	1.07	22.7	1.11	24.3	1.14
160	17.0	0.82	30.3	1.44	89.3	0.96	101.0	1.06	11.0	1.18	8.3	1.25	8.0	0.86	11.0	1.18	21.0	1.03	21.7	1.02
50	21.3	1.03	25.7	1.22	94.0	1.01	136.7	1.44	10.7	1.14	7.7	1.15	7.7	0.82	9.0	0.96	22.7	1.11	20.3	0.95
16	19.0	0.92	24.7	1.17	91.3	0.99	109.3	1.15	8.0	0.86	9.0	1.35	7.3	0.79	10.7	1.14	21.3	1.05	20.7	0.97
NPD (4mg)	316.7	15.32	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	833.3	10.00	–	–	767.3	92.08	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	428.0	45.86	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	537.3	31.61	–	–
2AA (2mg)	–	–	820.0	39.05	–	–	1164.7	12.26	–	–	126.7	19.00	–	–	174.3	18.68	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	145.3	6.81

Table 4 Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimurium tester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

22.3

1.29

25.0

1.10

88.3

0.87

117.0

1.09

12.7

1.15

11.7

1.17

8.0

0.92

10.0

1.30

18.0

1.04

26.0

1.24

DMSO Control

17.3

1.00

22.7

1.00

102.0

1.00

107.7

1.00

11.0

1.00

10.0

1.00

8.7

1.00

7.7

1.00

17.3

1.00

21.0

1.00

Ultrapure Water Control

–

–

–

–

95.0

1.00

–

–

11.0

1.00

–

–

–

–

–

–

23.3

1.00

–

–

5000

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

11.0

0.52

3000

–

–

0.0^A

0.00

–

–

0.0^A

0.00

–

–

–

–

–

–

–

–

12.0^SB

0.69

1600

–

–

10.0^B

0.44

–

–

0.0^B

0.00

–

–

0.0^B

0.00

–

–

0.0^B

0.00

14.7

0.85

29.3

1.40

1250

–

–

–

–

–

–

–

–

0.0^A

0.00

–

–

–

–

–

–

–

–

–

–

1000

1.0^B

0.06

–

–

0.0^A

0.00

–

–

–

–

–

–

0.0^A

0.00

–

–

–

–

–

–

750

–

–

–

–

–

–

–

–

0.0^B

0.00

–

–

–

–

–

–

–

–

–

–

500

9.0^SB

0.52

26.0

1.15

24.3^B

0.24

99.0

0.92

6.0^SB

0.55

7.0^SB

0.70

1.0^B

0.12

4.3^SB

0.57

20.3

1.17

30.3

1.44

160

11.7

0.67

20.0

0.88

53.7^SB

0.53

105.7

0.98

9.0

0.82

9.3

0.93

1.7^SB

0.19

7.3

0.96

18.7

1.08

29.3

1.40

50

17.7

1.02

25.3

1.12

103.0

1.01

109.3

1.02

10.3

0.94

10.0

1.00

9.0

1.04

7.0

0.91

20.0

1.15

31.0

1.48

16

19.0

1.10

19.3

0.85

98.7

0.97

101.0

0.94

9.0

0.82

9.0

0.90

7.3

0.85

6.3

0.83

18.3

1.06

37.7

1.79

5

18.7

1.08

–

–

97.7

0.96

–

–

9.0

0.82

10.0

1.00

7.7

0.88

9.3

1.22

–

–

–

–

NPD (4mg)

270.3

15.60

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

SAZ (2mg)

–

–

–

–

926.7

9.75

–

–

1397.3

127.03

–

–

–

–

–

–

–

–

–

–

9AA (50mg)

–

–

–

–

–

–

–

–

–

–

–

–

474.7

54.77

–

–

–

–

–

–

MMS (2mL)

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

900.0

38.57

–

–

2AA (2mg)

–

–

1058.7

46.71

–

–

976.0

9.07

–

–

122.3

12.23

–

–

87.3

11.39

–

–

–

–

2AA (50mg)

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

148.0

7.05

Obs: Observation                                                           

SD: Standard Deviation

MR: Mutation Rate

B: Reduced Background lawn development

SB: Slightly reduced Background lwan development

Conclusions:

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test substance is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

A Bacterial Reverse Mutation Assay (using Salmonella typhimurium and

Escherichia coli) with test item was conducted. The test item was dissolved in dimethyl sulfoxide (DMSO). In the Initial Mutation Test the following concentrations were examined:

 -S9 Mix: 3000, 1600, 500; 160; 50 and 16 μg/plate;

+S9 Mix: 5000; 1600, 500; 160; 50 and 16 μg/plate.

Because of the inhibitory, cytotoxic effect of the test item observed in the Initial Mutation Test, in the Confirmatory Mutation Test the concentration ranges were modified in the Salmonella typhimurium strains as follows:

in Salmonella typhimurium TA98 and TA100 strains:

-S9 Mix: 1000-5 μg/plate; +S9 Mix: 3000-16 μg/plate;

in Salmonella typhimurium TA1535 strain:

 S9 Mix: 1250-5 µg/plate; +S9 Mix: 1600-5 μg/plate;

and in Salmonella typhimurium TA1537 strain:

 S9 Mix: 1000-5 μg/plate; +S9 Mix: 1600-5 μg/plate.

 

In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (absent revertants, decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined bacterial strains. The 160 µg/plate was found to be the lowest cytotoxic concentration, observed in Confirmatory Mutation Test in the case of Salmonella typhimurium TA100 and TA1537 strains, in the absence of exogenous metabolic activation (S9 Mix). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-07-07 to 2016-09-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Principles of method if other than guideline:

There is a deviation from the guidelines regarding the confirmation of negative results. Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 28 July 2015).

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation test in vitro: HPRT assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) located at the X chromosome

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Suitability of cells: extensively validated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium containing 10 % fetal bovine serum; 5 % CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsome preparation (S9 Mix)

Test concentrations with justification for top dose:

Without S9 Mix:
110; 115; 120; 125; 130; 135 µg/mL

With S9:
20; 30; 40; 50; 60; 70 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: well solubility of test item, according to guideline

Untreated negative controls:

no

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:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10^6 per dish

DURATION
- Exposure duration: 5h
- Selection time (if incubation with a selection agent): 19 h
- Expression time (cells in growth medium): 8 d

SELECTION AGENT (mutation assays): hypoxanthine Ham's F12-SEL medium

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Rationale for test conditions:

Closely spaced concentrations (spacing factor < 2) were chosen as a steep dose-response curve was observed in the dose-range finding study. The concentrations selected covered a range from maximum (10-20% relative survival) to little or no cytotoxicity as recommended by Guideline.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

Test item 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:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (based 95% control limit).

The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:

- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 120 µg/mL onward (without S9) and from 60 µg/mL onward (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant changes in pH were found after treatment with the test item.
- Effects of osmolality: No relevant changes in osmolality were found after treatment with the test item.
- Precipitation: There was no precipitation observed in the medium at the end of treatment at any concentration tested.


RANGE-FINDING/SCREENING STUDIES: A Pre-test on Toxicity was performed to establish an appropriate concentration range for the main mutation assay, both in the absence and in the presence of metabolic activation (rodent S9-mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control.

HISTORICAL CONTROL DATA
- Positive historical control data: please refer to "Any other information on results"
- Negative (solvent/vehicle) historical control data: please refer to "Any other information on results"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: cloning efficiency
- Other observations when applicable: On Day 1, there was very clear evidence of toxicity with the test item in presence and absence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.

Table 1 Historical Background for Solvent Control Mutant Frequency (2015-2016)

	Without S9 Mix	With S9 Mix
	5-h exposure	5-h exposure
Mean	5.03	5.67
Sd	0.82	0.98
Range	2.00 - 8.82	1.96-11.76
Lower 95 % Confidence interval	3.18	3.47
Upper 95 % Confidence interval	6.88	7.88
n	10	10

 

Table 2 Historical Background for Positive Control Mutant Frequency (2015-2016)

	Without S9 Mix Ethyl methanesulphonate	With S9 Mix 7,12-Dimethylbenzanthracene
	5-h exposure	5-h exposure
Mean	1430.86	733.07
Sd	40.35	21.38
Range	951.06 – 1636.92	548.05 – 841.72
Lower 95 % Confidence interval	1339.58	684.72
Upper 95 % Confidence interval	1522.14	781.42
n	10	10

 

Table 3 CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-hour Treatment without s9-Mix)

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control a	200.7	±	2.08	100	100	1	0	0	4	2	7	101	6.93
Pos. control (EMS 1.0 µL/mL) a	52.3	±	2.08	26	63	190	180	191	202	200	963	64	1504.69**
TEST ITEM
110g/mL a	196.7	±	2.08	98	99	1	4	1	1	0	7	100	7.00
115g/mL a	183.3	±	2.52	91	99	4	2	0	0	0	6	99	6.06
120g/mL a	161.3	±	3.06	80	97	1	1	3	0	2	7	97	7.22
125g/mL a	71.7	±	1.53	36	98	1	0	1	3	1	6	98	6.12
130g/mL a	49.7	±	0.58	25	96	0	0	1	2	3	6	97	6.19
135g/mL a	29.0	±	1.00	14	97	0	2	1	1	2	6	98	6.12

 

Solvent control b	200.3	±	0.58	100	100	0	1	2	0	4	7	100	7.00
Pos. control (EMS 1.0 µL/mL) b	53.3	±	0.58	27	63	201	200	181	189	190	961	64	1501.56**
TEST ITEM
110g/mL b	196.7	±	1.15	98	99	0	0	3	1	2	6	99	6.06
115g/mL b	181.7	±	2.08	91	99	3	3	1	0	0	7	99	7.07
120g/mL b	160.3	±	1.53	80	97	1	0	2	3	1	7	97	7.22
125g/mL b	73.3	±	0.58	37	98	0	0	3	2	0	5	98	5.10
130g/mL b	51.0	±	1.00	25	98	0	4	1	1	0	6	98	6.12
135g/mL b	30.0	±	1.00	15	98	0	1	3	1	0	5	98	5.10

 

Solvent control c	201.3	±	1.15	100	100	1	1	3	1	2	8	101	7.92
Pos. control (EMS 1.0 µL/mL) c	52.7	±	0.58	26	62	193	195	183	191	187	949	62	1530.65**
TEST ITEM
110g/mL c	196.3	±	2.08	98	100	0	1	4	2	1	8	101	7.92
115g/mL c	182.0	±	1.73	90	95	0	3	3	0	0	6	96	6.25
120g/mL c	160.7	±	2.08	80	97	0	1	2	1	2	6	98	6.12
125g/mL c	72.0	±	1.00	36	96	0	1	0	0	4	5	97	5.15
130g/mL c	54.0	±	1.00	27	97	0	3	0	3	0	6	98	6.12
135g/mL c	30.3	±	1.53	15	98	3	2	1	1	0	7	99	7.07

 

Solvent control d	201.7	±	2.08	100	100	3	4	1	0	0	8	101	7.92
Pos. control (EMS 1.0 µL/mL) d	52.3	±	1.53	26	63	188	185	193	184	191	941	63	1493.65**
TEST ITEM
110g/mL d	197.0	±	2.00	98	99	0	1	6	1	0	8	100	8.00
115g/mL d	181.7	±	1.15	90	98	0	0	3	1	3	7	98	7.14
120g/mL d	161.0	±	1.00	80	98	0	3	1	1	3	8	98	8.16
125g/mL d	72.7	±	0.58	36	99	3	1	0	2	0	6	99	6.06
130g/mL d	54.0	±	1.00	27	98	0	4	3	0	0	7	98	7.14
135g/mL d	29.3	±	1.53	15	98	0	0	1	2	3	6	99	6.06

 

a, b, c, d = parallel of first culture.

abs.C.E. = Absolute Cloning Efficiency

EMS= Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control

Table 4 CHO/HPRT MUTAGENESIS ASSAY RESULTS MAIN MUTATION ASSAY/a, b, c and d (5-hour Treatment with s9-Mix)

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control a	200.3	±	0.58	100	100	0	1	3	2	3	9	100	9.00
Pos. control (DMBA 20 µg/mL) a	120.7	±	2.08	60	79	117	113	120	124	125	599	79	758.23**
TEST ITEM
20g/mL a	186.3	±	3.21	93	97	0	1	2	1	4	8	97	8.25
30g/mL a	165.7	±	2.31	83	99	2	0	1	3	1	7	99	7.07
40g/mL a	143.0	±	2.65	71	97	2	1	0	1	3	7	97	7.22
50g/mL a	118.7	±	2.08	59	96	0	1	3	2	2	8	96	8.33
60g/mL a	84.0	±	2.00	42	97	1	1	0	2	3	7	97	7.22
70g/mL a	32.7	±	1.15	16	97	3	0	3	0	2	8	97	8.25

 

Solvent control b	201.0	±	0.00	100	100	0	0	2	2	4	8	101	7.92
Pos. control (DMBA 20 µg/mL) b	122.0	±	1.00	61	79	123	127	119	122	126	617	80	771.25**
TEST ITEM
20g/mL b	186.7	±	1.53	93	97	0	1	0	2	4	7	97	7.22
30g/mL b	165.0	±	1.73	82	99	0	0	2	3	2	7	99	7.07
40g/mL b	144.3	±	1.15	72	97	0	1	1	3	1	6	98	6.12
50g/mL b	118.7	±	2.31	59	98	2	0	3	1	1	7	98	7.14
60g/mL b	85.0	±	1.00	42	97	1	0	0	3	3	7	98	7.14
70g/mL b	32.7	±	1.15	16	98	1	1	4	1	1	8	98	8.16

 

Solvent control c	200.0	±	1.00	100	100	1	0	1	2	4	8	100	8.00
Pos. control (DMBA 20 µg/mL) c	121.3	±	1.53	61	79	129	126	118	123	124	620	79	784.81**
TEST ITEM
20g/mL c	184.3	±	2.08	92	97	0	1	3	0	4	8	97	8.25
30g/mL c	168.0	±	2.00	84	98	1	5	0	1	0	7	98	7.14
40g/mL c	140.0	±	1.73	70	96	2	0	0	4	0	6	96	6.25
50g/mL c	119.7	±	2.08	60	96	1	0	2	2	2	7	96	7.29
60g/mL c	84.0	±	2.00	42	97	2	3	2	0	0	7	97	7.23
70g/mL c	30.3	±	0.58	15	97	2	0	2	2	2	8	97	8.25

 

Solvent control d	201.0	±	1.0	100	100	5	2	0	1	0	8	101	7.92
Pos. control (DMBA 20 µg/mL) d	123.7	±	1.53	62	79	123	119	128	120	120	610	80	762.50**
TEST ITEM
20g/mL d	184.0	±	1.73	92	98	0	0	5	2	0	7	98	7.14
30g/mL d	167.0	±	1.00	83	97	6	0	1	0	0	7	98	7.14
40g/mL d	142.3	±	2.08	71	98	3	0	0	3	0	6	98	6.12
50g/mL d	118.7	±	0.58	59	97	2	0	3	1	1	7	97	7.22
60g/mL d	82.3	±	1.53	41	97	3	2	0	0	2	7	98	7.14
70g/mL d	31.3	±	1.53	16	97	4	1	2	0	1	8	98	8.16

 

a, b, c, d = parallel of first culture.

abs.C.E. = Absolute Cloning Efficiency

EMS= Ethyl methanesulfonate

** = p < 0.01 to the concurrent negative control and to the historical control

 

Conclusions:

The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells.
Thus, the test item was not mutagenic under the conditions of this study.

Executive summary:

The test item, dissolved in Dimethyl sulfoxide (DMSO), was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital andβ-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix:

110, 115, 120, 125, 130 and 135 μg/mL

5-hour treatment period with S9-mix:

20, 30, 40, 50, 60 and 70 μg/mL

In the performed Mutation Assay the concentration levels were chosen based on the cytotoxicity.

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The validity of the test and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL). The mutation frequency found in the positive controls was within the range of historical laboratory control data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation test:

A Bacterial Reverse Mutation Assay (using Salmonella typhimurium and Escherichia coli) with test item was conducted. The test item was dissolved in dimethylsulfoxide (DMSO). In the Initial Mutation Test the following concentrations were examined:

 -S9 Mix: 3000, 1600, 500; 160; 50 and 16 μg/plate;

+S9 Mix: 5000; 1600, 500; 160; 50 and 16 μg/plate.

Because of the inhibitory, cytotoxic effect of the test item observed in the Initial Mutation Test, in the Confirmatory Mutation Test the concentration ranges were modified in the Salmonella typhimurium strains as follows:

in Salmonella typhimurium TA98 and TA100 strains:

-S9 Mix: 1000-5 μg/plate; +S9 Mix: 3000-16 μg/plate;

in Salmonella typhimurium TA1535 strain:

 S9 Mix: 1250-5 µg/plate; +S9 Mix: 1600-5 μg/plate;

and in Salmonella typhimurium TA1537 strain:

 S9 Mix: 1000-5 μg/plate; +S9 Mix: 1600-5 μg/plate.

 

In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. Five bacterial strains were used to investigate the mutagenic potential of the test item in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. In the performed experiments all of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effect of the test item (absent revertants, decreased number of revertant colony numbers and/or affected background lawn development) was observed in all examined bacterial strains. The 160 µg/plate was found to be the lowest cytotoxic concentration, observed in Confirmatory Mutation Test in the case of Salmonella typhimurium TA100 and TA1537 strains, in the absence of exogenous metabolic activation (S9 Mix). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

In vitro mammalian chromosome aberration test:

The test item, was tested in a Chromosome Aberration Assay in V79 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 50, 100, 125 and 1501 μg/mL

with S9 mix: 100, 150, 175 and 200 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 12.5, 25, 50 and 751 μg/mL

Experiment B with 20/28 h treatment/sampling time without S9 mix: 12.5, 25, 50 and 75 1 μg/mL

Experiment B with 3/28 h treatment/sampling time with S9 mix: 100, 150, 175 and 200 μg/mL

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted. In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to cytotoxic concentrations in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations. In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberration rates were within the ranges of historical control data. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test was shown as the concurrent positive controls Ethyl methanesulfonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) caused the expected increases in cells with structural chromosome aberrations and were compatible with the historical control range.

In vitro mammalian gene mutation assay:

The test item, dissolved in Dimethyl sulfoxide (DMSO), was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital andβ-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix:

110, 115, 120, 125, 130 and 135 μg/mL

5-hour treatment period with S9-mix:

20, 30, 40, 50, 60 and 70 μg/mL

In the performed Mutation Assay the concentration levels were chosen based on the cytotoxicity.

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The validity of the test and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL). The mutation frequency found in the positive controls was within the range of historical laboratory control data.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.