Bis(2-(2-butoxyethoxy)ethyl) adipate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Mutagenicity (OECD 471, GLP), Ames: negative with and without metabolic activation

Cytogenicity/micronucleus test in peripheral human lymphocytes (OECD 487, GLP): negative with and without metabolic activation

 

Gene mutation in mammalian cells (OECD 490, GLP): negative with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July to 17 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (Salmonella typhimurium strains); trp operon (E.coli strains)

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1 and 2
50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthtracene (2AA)

Remarks:

+S9: 2AA (all strains); -S9: N-ethyl-N-nitro-N-nitrosoguanidine (ENNG), benzo(a)pyrene (BP), 9-aminoacridine (9AA), 4-nitroquinoline-N-oxide (4NQO)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Standard plate incorporation method (experiment 1) and preincubation method (experiment 2)

DURATION
- Preincubation period: 20 min (only exp 2)
- Exposure duration: 48 h at 37 °C (exp 1 and 2)

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.

Evaluation criteria:

Acceptance Criteria:

The following criteria must be met for acceptance:
- All tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls.
- The appropriate characteristics of each tester strain must be confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor.
- All tester strain cultures should be in the range of 0.9 to 9.9 x 10^9 bacteria per mL.
- Each mean positive control value should be at least 2x the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains and the integrity of the S9-mix
- The test should include a minimum of four non-toxic dose levels.

Evaluation Criteria:

There are several criteria for determining a positive result. Any, one, or all of the following may be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentrations.
- The biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by the UKEMS (Mahon et al., 1989).
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Mean values with standard deviations were calculated.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDIES
In order to select appropriate dose levels for use in the main test, a preliminary test were conducted in the presence or absence of metabolic activation. Ten dose levels and controls were tested up to and including 5000 µg/plate at approximately half-log intervals. The assay was conducted by mixing 0.1 mL the bacterial culture (TA 100 or WP2uvrA-), and 0.1 mL of the vehicle or test chemical mixture, 0.5 mL of S9-mix or phosphate buffer and 2.0 mL of molten agar supplemented with trace histidine or tryptophan and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 °C, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Numbers of revertant colonies in the preliminary toxicity test

With (+) or without (-) S9-mix	Strain	Dose (µ/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA 100	90	88	99	108	113	114	107	86	114	118	113*
+	TA 100	120	112	108	121	100	98	92	96	90	98	105*
-	WP2 uvrA-	43	40	40	43	48	42	38	38	45	41	42*
+	WP2 uvrA-	47	43	48	43	38	37	42	41	37	38	42*

* Precipitate present

Table 2: Range-finding test without metabolic activation

	Revertant colony counts (mean of 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	105	19	28	24	13
50	111	16	24	25	10
150	120	14	28	29	12
500	105	15	31	25	11
1500	109P	17P	26P	28P	12P
5000	115P	14P	30P	24P	13P
ENNG (3)	497
ENNG (5)		162
ENNG (2)			283
4NQO (0.2)				116
9AA (80)					2530

P - precipitate

Abbreviations: ENNG, N-ethyl-N’-nitro-N-nitrosoguanidine; 9AA, 2-aminoacridine; BP, benzo(a)pyrene; 2AA, 2-aminoanthracene; 4NQO, 4- nitroquinoline-1-oxide

Table 3: Range-finding test with metabolic activation

	Revertant colony counts (mean 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	115	14	33	29	12
50	98	13	32	27	12
150	109	16	32	30	11
500	99	10	29	29	12
1500	115P	14P	31P	29P	12P
5000	116P	14P	32P	29P	14P
2AA (1)	801
2AA (2)		292			95
2AA (10)			130
BP (5)				189

P - preicipitate

Table 4: Main Test without metabolic activation

	Revertant colony counts (mean 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	117	21	26	34	9
50	115	20	35	27	11
150	117	20	30	27	9
500	115	21	31	30	10
1500	117P	20P	31P	31P	11P
5000	124P	18P	30P	32P	7P
ENNG (3)	439
ENNG (5)		174
ENNG (2)			182
4NQO (0.2)				122
9AA (80)					730

P - precipitate.

Table 5: Main Test with metabolic activation

	Revertant colony counts (mean 3 replicates)
Test substance concentration (µg/plate)	TA 100	TA 1535	WP2 uvrA-	TA98	TA1537
0	124	24	41	29	13
50	117	18	38	24	10
150	127	18	36	30	9
500	119	11	34	33	12
1500	114P	13P	29P	26P	10P
5000	130P	18P	35P	22P	9P
2AA (1)	952
2AA (2)		261			217
2AA (10)			202
BP (5)				309

P - precipitate

Conclusions:

Interpretation of results (migrated information):
negative

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May - 20 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: blood was collected from healthy adult, non-smoking volunteers
- Suitability of cells: peripheral human lymphocytes are recommended in the international OECD guideline 487
- Normal cell cycle time (negative control): average generation times were determined to be 14,8 h, 14.4 h, 13.9 h and 14.2 h for the respective donors (December 2017)

For lymphocytes:
- Age and number of blood donors: 23, 26 or 32 years, 4
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not pooled
- Mitogen used for lymphocytes: 0.1 mL (9 mg/mL) phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine,(2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands); 5.0 ± 0.5% CO2, 80 - 100% (humidity), 37.0 ± 1.0°C

Cytokinesis block (if used):

Cytochalasine B (Sigma; 5 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : rat S9 homogenate (Trinova Biochem GmbH, Giessen, Germany) was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life Technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin)
- concentration or volume of S9 mix and S9 in the final culture medium: 1.8% (v/v)

Test concentrations with justification for top dose:

Dose-range finding test: 31.3, 62.5, 125, 250, 500 and 1000 μg/mL (+/- S9, 3h exposure) and 62.5, 125, 250, 500, 1000 and 2000 μg/mL (- S9, 24h exposure). Precipitation was observed at dose levels of 125 μg/ml and upwards after adding the test item to the culture medium. However, the amount of precipitate decreased during incubation. After 3 h the precipitate was only observed at dose levels of 1000 μg/mL and upwards.

First cytogenetic assay: 50, 500, 750 and 1000 μg/mL (+/-S9, 3 h exposure, 27 h harvest time). Based on precipitates observed in the dose range finding experiment. Due to an error 2 dose levels were selected with precipitate, which is not according to OECD 487. Since the amount of precipitate did not interfere with the scoring and clear results were obtained this does not impact the validity of the study. In the presence of S9-mix the positive control did not give a proper response.

Therefore, the experiment was rejected and repeated in cytogenetic assay 1A with the following dose levels: 50, 500 and 750 μg/mL culture medium (+/-S9, 3 h exposure, 27 h harvest time), In this repeat experiment only one dose level was tested with precipitate.

Second cytogenetic assay: 50, 500, 750 and 1000 μg/mL culture medium (- S9, 24 h exposure, 24 h harvest time). Based on precipitates observed in the dose range finding experiment.

Vehicle / solvent:

- Vehicle used: DMSO (Merck, Darmstadt, Germany)
- Justification for choice of vehicle: a solubility test was performed based on visual assessment. The test item formed a clear colourless solution in dimethyl sulfoxide.
- Justification for percentage of vehicle in the final culture medium: 1.0% (v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments 3

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium (0.4 mL blood was added to 5 mL or 4.8 mL culture medium, +/- S9, respectively and 0.1 mL (9 mg/mL) phytohaemagglutinin)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 46 ± 2 h
- Exposure duration/duration of treatment: 3h / 24h
- Harvest time after the end of treatment: 24 h

FOR MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 0.1 μg/mL (3 h exposure) and 0.05 μg/mL (24 h exposure) colchicin was used as a direct acting aneugen.
- If cytokinesis blocked method was used for micronucleus assay: cytochalasine B (Sigma; 5 μg/mL) was added to the culture after the end of the 3 h expsore period for 24 h (first cytogenetic assay +/- S9) or cytochalasin B (5 μg/mL) was added during the test substance exposure period to the medium for 24 h (second cytogenetic assay, -S9).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): fixed slides were stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8.
- Number of cells spread and analysed per concentration: at least 2000 (with a maximum deviation of 5%) binucleated cells per concentration
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
The following criteria for scoring of binucleated cells were used (1 - 2, 6):
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytostasis / cytotoxicity was determined by determination of cytokinesis-Block Proliferation Index (CBPI)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
At least 2000 binucleated cells per concentration were examined by light microscopy for micronuclei. In addition, at least 2000 mononucleated cells per concentration were scored for micronuclei separately. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000.

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes:

Remarks:

peripheral human (3h exposure)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes:

Remarks:

peripheral human (24 h exposure)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

53% and 59% cytostatisis (CBPI) was reached at 750 and 1000 μg/mL, respectively. Precipitate was observed at the two highest dose levels (750 and 1000 μg/ml).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 8.0 at 62 µg/mL (compared to 8.1 in the vehicle control). Since pH and osmolarity detmerination is performed at the beginning of the assay, 62.5 μg/ml was the highest non precipitating dose level, and measurements were performed at this concentration.
- Data on osmolality: 436 mOsm/kg (compared 439 mOsm/kg in the vehicle control)
- Precipitation and time of the determination:
Dose range finding test: Precipitates were observed at a concentration of 125 μg/mL and upwards after adding the test item to the culture medium. After 3h precipitate was only observed at dose levels of 750 μg/mL and upwards. Precipitates were visible at 1000 µg/mL (3h exposure, +/- S9) and 1000 and 2000 µg/mL (24 h exposure, -S9)
First cytogenetic assay: Precipitates were observed at at the two highest dose levels 750 and 1000 µg/mL (3h exposure, +/- S9) --> positive control did not give a proper response, therefore this experiment was rejected
First cytogenetic assay (1A): Precipitates were observed at the highest dose level of 750 µg/mL (3h exposure, +/- S9)
Secon cytogenetic assay: Precipitates were observed at the two highest dose levels 750 and 1000 µg/mL (24 h exposure, -S9)
- Definition of acceptable cells for analysis: 2000 cells (binucleated) / concentration
Other: First cytogenetic assay: Positive control did not give a proper response, therefore this experiment was rejected.

RANGE-FINDING/SCREENING STUDIES: a preliminary assay with 3h (+/- S9) and 24 h (-S9) exposure was performed up to an concentration of 1000/2000 µg/mL, respectively. Precipitates were observed at a concentration of 125 μg/mL and upwards after adding the test item to the culture medium. However, the amount of precipitate decreased during incubation. After 3 h the precipitate was only observed at dose levels of 750 μg/mL and upwards. Substantial toxicity (at least 55 ± 5 % cytostasisl were observed at 1000 µg/mL (3h exposure and 24 h exposure -S9) and 2000 µg/mL (24 h exposure -S9). Please refer to table 1 in the " any other information on results including tables section".

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The number of mono- and binucleated cells with micronuclei found in the vehicle control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced
a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95%
control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

For all test methods and criteria for data analysis and interpretation:
- ACCEPTABILITY CRITERIA
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with
micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).
d) Cell proliferation criteria in the solvent control are fulfilled to assure that sufficient treated cells have undergone mitosis during the test and that the treatments are conducted at appropriate levels of cytotoxicity.
e) All experimental conditions are tested unless one resulted in positive results.
f) Adequate number of cells and concentrations were analyzed.
g) The criteria for the selection of the top concentration are fulfilled.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: Substantial toxicity (at least 55 ± 5 % cytostasisl) was not observed in the first cytogenetic experiment (1A) . In the second cytogenetic assay toxicity, cytokinesis-block proliferation index of 55 ± 5% was determined at 750 and 1000 µg/mL.

- Genotoxicity results
- The test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

HISTORICAL CONTROL DATA
- Positive historical control data: Mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. Colchicine produced a statistically significant increase in the
number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Please refer to table 13 in the " any other information on results including tables section".
- Negative (vehicle) historical control data: The number of mono- and binucleated cells with micronuclei found in the vehicle control was within the 95% control limits of the distribution of the historical negative control database. Please refer to table 12 in the " any other information on results including tables section".

Remarks on result:

other: Under the conditions of the conducted test, the test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9.

Table 1: Cytokinesis-block proliferation index of human lymphocyte cultures in the dose-range finding test

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Number of cells with ….nuclei			CBPI	% cytostasis
	1	2	3 or more
0 (vehicle)	325	169	11	1.38	0
31.3	328	159	13	1.37	2
62.5	368	124	8	1.28	26
125	365	125	10	1.29	23
250	365	133	2	1.27	28
500	392	111	4	1.23	38
1000^1)	417	80	3	1.17	55
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Number of cells with ….nuclei			CBPI	% cytostasis
	1	2	3 or more
0 (vehicle)	328	157	15	1.37	0
31.3	342	147	11	1.34	10
62.5	341	144	15	1.35	7
125	363	132	5	1.28	24
250	381	112	8	1.26	32
500	385	107	8	1.25	34
1000^1)	399	98	3	1.21	44
With metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/mL	Number of cells with ….nuclei			CBPI	% cytostasis
	1	2	3 or more
0 (vehicle)	327	155	18	1.38	0
62.5	364	128	8	1.29	25
125	347	156	9	1.34	11
250	331	162	7	1.35	8
500	383	117	0	1.23	39
1000^1)	409	91	0	1.18	52
2000^1)	484	16	0	1.03	92

Note: All calculations were performed without rounding off.

¹⁾The test item precipitated in the culture medium

 

Table 2: Cytokinesis-block proliferation index of human lymphocytes cultures in the first cytogenetic assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI	% cytostasis
0 (vehicle)	1.27	-	1.45	1.36	0
50	1.24	-	1.25	1.25	31
500	1.24	-	1.28	1.26	27
750^1)	1.27	-	1.29	1.28	22
1000^1)	1.24	-	1.27	1.26	29
0.25 MMC-C	1.20	-	1.21	1.20	43
0.38 MMC-C	1.18	-	1.20	1.19	47
0.1 Colchicin	1.04	-	1.04	1.04	90
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			CBPI	% cytostasis
0 (vehicle)	1.35	-	1.36	1.36	0
50	1.30	-	1.31	1.31	14
500	1.28	-	1.38	1.33	7
750^1)	1.28	-	1.35	1.31	12
1000^1)	1.29	-	1.30	1.30	17
15 CP	1.24	-	1.37	1.30	15
15 CP	1.32	-	1.33	1.33	9

Note: All calculations were performed without rounding off. CP:CyclophosphamideMMC-C: Mitomycin C

¹⁾The test item precipitated in the culture medium

 

Table 3: Cytokinesis-block proliferation index of human lymphocytes cultures in cytogenetic assay 1a

With metabolic activation (+ S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI	Mean CBPI	% cytostasis
0 (vehicle)	1.27 - 1.42	1.35	0
50	1.39 -1.39	1.39	-13
500	1.25 - 1.27	1.26	24
750^1)	1.21 - 1.32	1.27	22
15 CP	1.13 - 1.16	1.15	58
17.5 CP	1.10 - 1.14	1.12	65

Note: All calculations were performed without rounding off. CP:Cyclophosphamide

¹⁾The test item precipitated in the culture medium

 

Table 4: Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures in the first cytogenetic assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei 1)			Number of binucleated cells with micronuclei 1)
		1000	1000	2000	1000	1000	2000
		A	B	A+B	A	B	A+B
0 (vehicle)	0	0	0	0	0	3	3
500	27	0	0	0	1	3	4
750	22	0	0	0	0	2	2
1000	29	0	0	0	2	2	4
0.25-C	43	0	1	1	7	7	14**
0.38-C	47	1	0	1	13	9	22***
0.1 Colch	90	28	20	48***	36	42	78***

CP:CyclophosphamideMMC-C: Mitomycin C

 

Table 5: Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures in cytogenetic assay 1a

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000	1000	2000
		A	B	A+B	A	B	A+B
0 (vehicle)	0	0	0	0	2	0	2
50	-13	1	0	1	2	1	3
500	24	0	0	0	0	2	2
750	22	0	0	0	2	1	3
15 CP	58	1	2	3*	20	15	35***

CP:CyclophosphamideMMC-C: Mitomycin C

*)          Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

¹⁾           1000 bi- and mononucleated cells were scored for the presence of micronuclei.

              Duplicate cultures are indicated by A and B.

 

Table 6: Cytokinesis-block proliferation index of human lymphocyte cultures in the second cytogenetic assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI	% cytostasis
0 (vehicle)	1.34	-	1.34	1.34	0
50	1.31	-	1.32	1.32	7
500	1.25	-	1.30	1.28	19
750^1)	1.15	-	1.17	1.16	53
1000^1)	1.14	-	1.14	1.14	59
0.15 MMC-C	1.12	-	1.14	1.13	62
0.23 MMC-C	1.11	-	1.11	1.11	68
0.05 Colchicin	1.00	-	1.01	1.00	99

CP:CyclophosphamideMMC-C: Mitomycin C Note: All calculations were performed without rounding off.

¹⁾The test item precipitated in the culture medium

 

Table 7: Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures in the second cytogenetic assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei 1)			Number of binucleated cells with micronuclei 1)
		1000	1000	2000	1000	1000	2000
		A	B	A+B	A	B	A+B
0 (vehicle)	0	2	1	3	2	2	4
50	7	2	0	2	2	3	5
500	19	1	0	1	0	4	4
750	53	1	2	3	4	4	8
0.15-C	62	1	1	2	19	17	36***
0.05 Colchicin	99	18	20	38***	2^2)	1^2)	3

CP:CyclophosphamideMMC-C: Mitomycin C

*)          Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1)          1000 bi- and mononucleated cells were scored for the presence of micronuclei.

              Duplicate cultures are indicated by A and B.

2)          365 and 275 binucleated cells were scored for the presence of micronuclei, respectively. 

 

Table 8: Scoring of cells with one, two or more nuclei of human lymphocyte cultures in the first cytogenetic assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Culture	Number of cells with ….nuclei			CBPI
		1	2	3 or more
0 (vehicle)	A	375	117	8	1.27
	B	280	213	7	1.45
50	A	382	115	3	1.24
	B	378	117	5	1.25
500	A	380	118	2	1.24
	B	365	130	5	1.28
750	A	364	135	1	1.27
	B	357	141	2	1.29
1000	A	381	119	0	1.24
	B	364	136	0	1.27
0.25 MMC-C	A	399	101	0	1.20
	B	397	103	0	1.21
0.38 MMC-C	A	408	92	0	1.18
	B	400	100	0	1.20
0.1 Colch	A	484	14	2	1.04
	B	483	16	1	1.04
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Culture	Number of cells with ….nuclei			CBPI
		1	2	3 or more
0 (vehicle)	A	336	166	7	1.35
	B	324	171	5	1.36
50	A	348	147	5	1.31
	B	351	146	3	1.30
500	A	359	140	1	1.28
	B	313	184	3	1.38
750	A	365	132	3	1.28
	B	328	168	4	1.35
1000	A	353	147	0	1.29
	B	352	147	1	1.30
15 CP	A	385	112	3	1.24
	B	316	183	1	1.37
17.5 CP	A	338	160	2	1.33
	B	342	155	3	1.32

CP:CyclophosphamideMMC-C: Mitomycin C

 

Table 9: Scoring of cells with one, two or more nuclei of human lymphocyte cultures in cytogenetic assay 1a

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	Culture	Number of cells with ….nuclei			CBPI
		1	2	3 or more
0 (vehicle)	A	368	127	5	1.27
	B	298	196	6	1.42
50	A	324	188	7	1.39
	B	312	182	6	1.39
500	A	368	129	3	1.27
	B	381	111	8	1.25
750	A	399	95	6	1.21
	B	344	151	5	1.32
15 CP	A	419	81	0	1.16
	B	436	64	0	1.13
17.5 CP	A	430	70	0	1.14
	B	450	50	0	1.10

CP:CyclophosphamideMMC-C: Mitomycin C

 

Table 10: Scoring of cells with one, two or more nuclei of human lymphocyte cultures in the second cytogenetic assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/mL	Culture	Number of cells with ….nuclei			CBPI
		1	2	3 or more
0 (vehicle)	A	338	154	8	1.34
	B	344	141	15	1.34
50	A	347	145	8	1.32
	B	351	141	8	1.31
500	A	356	139	5	1.30
	B	378	117	5	1.25
750	A	425	75	0	1.15
	B	417	81	2	1.17
1000	A	430	69	1	1.14
	B	433	66	1	1.14
0.15 MMC-C	A	433	66	1	1.14
	B	438	62	0	1.12
0.23 MMC-C	A	446	54	0	1.11
	B	446	54	0	1.11
0.05 Colchicin	A	497	3	0	1.01
	B	499	1	0	1.00

CP:CyclophosphamideMMC-C: Mitomycin C

 

Table 11: Total number of cells with micronuclei; treatment/control comparison (mononucleated/binucleated cells)¹⁾

Exposure	S9-Mix	Nucleated	p-value	Decision at 95%
Dose µg/mL			one-sided	confidence interval
First cytogenetic assay (Chi-square test)
3 h exposure
MMC-C (0.25)	-	bi	= 0.0038	significant
MMC-C (0.38)	-	bi	<0.0001	significant
Colch (0.1)	-	mono	<0.0001	significant
Colch (0.1)	-	bi	<0.0001	significant
CP (15)	+	mono	= 0.0416	significant
CP (15)	+	bi	<0.0001	significant
Second cytogenetic assay (Chi-square test)
24 h exposure
MMC-C (0.15)	-	bi	<0.0001	significant
Colchicin (0.05)	-	mono	<0.0001	significant

1) Only statistically significant results are presented CP:CyclophosphamideMMC-C: Mitomycin C

 

Table 12: Historical control data for in vitro micronucleus studies of the solvent control

	Mononucleated			Binucleated
	+ S9-mix	- S9-mix		+ S9-mix	- S9-mix
	3 hour exposure	3 hour exposure	24 hour exposure	3 hour exposure	3 hour exposure	24 hour exposure
Mean number of micronucleated cells (per 1000 cells)	0.83	1.00	0.86	3.67	3.57	3.58
SD	0.95	1.01	1.12	2.53	2.20	2.51
n	112	114	110	112	114	110
Upper control limit (95% control limits)	2.85	3.37	3.60	9.42	8.94	9.64
Lower control limit (95% control limits)	-1.19	-1.38	-1.87	-2.08	-1.79	-2.48

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments using different vehicles e.g. DMSO, however these vehicles never influence the number of micronucleated cells) between October 2014 and October 2017.

 

Table 13: Historical Control Data for in vitro Micronucleus Studies of the Positive Control Substances

	Mononucleated		Binucleated
	- S9-mix		+ S9-mix	- S9-mix
	3 hour exposure (Colchicin)	24 hour exposure (Colchicin)	3 hour exposure (CP)	3 hour exposure (MMC-C)	24 hour exposure (MMC-C)
Mean number of micronucleated cells (per 1000 cells)	15.51	17.54	20.40	24.90	24.59
SD	16.52	21.30	8.54	20.69	22.65
n	234	230	122	234	230
Upper control limit (95% control limits)	57.06	65.59	37.20	59.48	69.14
Lower control limit (95% control limits)	-26.03	-30.51	3.59	-9.68	-19.97

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between October 2014 and October 2017. CP:CyclophosphamideMMC-C: Mitomycin C

 

Conclusions:

Under the conditions of the conducted test, the test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 March - 30 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Target gene:

thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: American Type Culture Collection, (ATCC, Manassas, USA, 2001)
- Suitability of cells: recommended cell line according to OECD TG 490

For cell lines:
- Absence of Mycoplasma contamination: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: 5.0 ± 0.5% CO2, humid atmosphere (80 - 100%, actual range 24 - 98%), 37.0 ± 1.0°C (actual range 34.3 - 38.5 °C).

Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).

Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

Exposure medium
For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT) (Sigma).

Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).

Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes (S9 homogenate) (Trinova Biochem GmbH, Giessen, Germany) and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies). The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration of S9 mix in the final culture medium: 4%

Test concentrations with justification for top dose:

Dose-range-finding test (3 h and 24 h exposure): 63, 125, 250, 500, 600, 700, 800, 900 and 1000 μg/mL (3h, +/- S9 and 24h, -S9). The test item precipitated in the exposure medium at concentrations of 1000 μg/mL and
above. Therefore, 1000 µg/mL was used as the highest test item concentration in the dose range finding test.
Cytotoxicity, determined by relative suspension growth was 74% at 500 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 1000 μg/mL (3h, - S9). No toxicity in the relative suspension growth was observed up to test item concentrations of 1000 μg/mL compared to the vehicle control (3h, +S9). The relative suspension growth was 37% at the test item concentration of 1000 μg/mL compared to the relative suspension growth of the vehicle control (24h, -S 9).

First mutagenicity test (3 h exposure): 16, 31, 63, 125, 250, 500, 600, 700, 800, 900 and 1000 μg/mL (- S9) and 7.8, 16, 31, 63,125, 250, 500 and 1000 μg/mL (+ S9). Doses selected based on results of the dose-range-finding test.

Second mutagenicity test (24 h exposure): 7.8, 16, 31, 63, 125, 250, 500 and 700 μg/mL (- S9). Doses selected based on results of the dose-range-finding test.

Vehicle / solvent:

- Vehicle used: DMSO

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in dimethyl sulfoxide.

- Justification for percentage of solvent in the final culture medium: 1% (v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 5 wells with mutants per selection plate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3h/24h

FOR GENE MUTATION:
- Expression time: 2 days
- Selection time: 11/12 days
- Method used: microwell plates
- Selective agent used: 5 μg/mL trifluorothymidine (Sigma)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates for determination of mutation frequency
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG) / relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Increase in mutation frequency

Evaluation criteria:

- For the micro well version of the assay the GEF is 126 x 10-6.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

3 h exposure

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

RTG (%) was 88, 90, 38 and 20 at concentrations of 500, 600, 700 and 800 µg/mL, resepctively (-S9) and 89, 93 and 82 at concentrations of 250, 500 and 1000 µg/mL, resepctively (+S9). Precipitates: 700 and 800 µg/mL (-S9) and 1000 µg/mL (+S9).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

24 h exposure

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

RTG% was 74, 77 and 52 at concentrations of 250, 500 and 700 µg/mL, respectively. Precipitates: 700 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated in the exposure medium at concentrations of 700 and 800 µg/mL (3 h, - S9), 1000 µg/mL (3 h, +S9) and 700 µg/mL (24 h, -S9)

RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test, L5178Y mouse lymphoma cells were treated with the test item (concentration range of 63 to 1000 μg/mL) in the absence of S9-mix with 3 and 24 h exposure and in the presence of S9-mix with a 3 h exposure. The test item precipitated in the exposure medium at concentrations of 1000 μg/mL and above. Therefore, 1000 µg/mL was used as the highest test item concentration in the dose range finding test. Cytotoxicity, determined by relative suspension growth was 74% at 500 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 1000 μg/mL (3h, - S9). No toxicity in the relative suspension growth was observed up to test item concentrations of 1000 μg/mL compared to the vehicle control (3h, +S9). The relative suspension growth was 37% at the test item concentration of 1000 μg/mL compared to the relative suspension growth of the vehicle control (24h, -S 9).

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The mutation frequency found in the vehiclet control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical
negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

For all test methods and criteria for data analysis and interpretation:
- Any other criteria: For the micro well version of the assay the GEF is 126 x 10-6.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10-6).
e) The upper limit of cytotoxicity observed in the positive control culture should be the same as of the experimental cultures. That is, the RTG/RS should not be less than 10%.The MF of the positive control must be within the acceptable range established for the laboratory
f) Two experimental conditions (short treatment with and without metabolic activation) were conducted unless one resulted in positive results.
g) Adequate number of cells and concentrations should be analysable
h) The criteria for the selection of top concentration are consistent with those described in OECD Guideline 490

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: First mutagenicity test: RTG (%) was 88, 90, 38 and 20 at concentrations of 500, 600, 700 and 800 µg/mL, resepctively (-S9) and 89, 93 and 82 at concentrations of 250, 500 and 1000 µg/mL, resepctively (+S9). Dose levels of 16 to 250 μg/mL showed no cytotoxicity (3h, -S9). The dose levels of 900 and 1000 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. Second mutagenicity test: RTG% was 74, 77 and 52 at concentrations of 250, 500 and 700 µg/mL, respectively.

- Genotoxicity results:
o Concentrations selected for evaluation based on toxicity: First mutagenicity test: 31, 63, 125, 250, 500, 600, 700 and 800 μg/mL (3h, -S9), 7.8, 16, 31, 63, 125, 250, 500 and 1000 μg/mL (3h, +S9) In the presence of S9-mix, no significant toxicity was observed and all dose levels were evaluated. Second mutagenicity test: 7.8, 16, 31, 63, 125, 250, 500, 600 and 700 μg/mL (24h, -S9). No severe toxicity was observed up to and including the highest tested dose level and all dose levels were evaluated
o Number of cells treated and sub-cultures for each cultures : Per culture 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) (Treatment of cells)
o Number of cells plated in selective and non-selective medium : 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium

HISTORICAL CONTROL DATA
- Positive historical control data: Please refer to table 9 in the " any other information on results including tables section".
- Negative (vehicle) historical control data: Please refer to table 8 in the " any other information on results including tables section".

Table 1: Dose-range finding test: Cytotoxicity of the test item (3 h and 24 treatment)

Dose	cell count after 24 h of subculture (cells/ml x10^5)	cell count after 48 h of subculture (cells/ml x10^5)	SG^(1)	RSG^(2)
(μg/ml)				(%)
3h, without metabolic activation
SC	5.2	7.3	19	100
63	5.8	6.8	20	103
125	5.3	7.1	19	98
250	5.6	7.0	20	103
500	4.3	6.5	14	74
1000^(3)	0.1^(4)	0.4	0	0
3h, with metabolic activation
SC	3.7	5.4	10	100
63	3.5	6.9	12	121
125	2.3	5.8	7	67
250	4.0	6.1	12	121
500	4.6	5.4	12	123
1000^(3)	3.5	5.7	10	100
24h, without metabolic activation
SC	11.1	5.7	41	100
63	11.5	6.2	45	112
125	10.6	6.3	43	105
250	9.3	6.3	38	93
500	8.8	6.2	35	86
1000^(3)	3.7	6.3	15	37

Note: all calculations were made without rounding off

SC = solvent control = DMSO

 

(1) = suspension growth

(2) = relative suspension growth

(3) = the test item precipitated in the exposure medium

(4) = since less than 1.25 x 105 c/ml were present, no subculture was performed

 

3h exposure:SG = Suspension growth = [Cell count after 24 hour of subculture (Day 1) /1.6 x 105] x [Cell count after 48 hours of subculture (Day 2)/1.25 x 105]*

 

24 h exposure: SG = Suspension growth = [Day 0 cell count/1.25 x 105 ] x [Day 1 cell count/1.25 x 105 ]

 

* Or appropriate cell concentration

RSG = [SG(test)/SG(control)] x 100

 

Table 2: Experiment 1: Cytotoxic and mutagenic response of the test substance in the mouse lymphoma l5178Y test system

Dose	RSG	CE day2	RCE	RTG	Mutation frequency per 10^6survivors
(µg/mL)	(%)	(%)	(%)	(%)	total	small	large
3h exposure, without metabolic activation
SC1	100	72	100	100	100	57	40
SC2		69			144	59	78
31	89	85	120	107	66	30	34
63	102	52	74	75	106	49	53
125	90	76	107	96	139	79	53
250	91	94	133	121	90	47	39
500	78	80	113	88	128	56	66
600	68	94	133	90	123	67	49
700^(1)	36	76	107	38	135	80	48
800^(1)	17	84	118	20	121	64	51
MMS	87	51	72	63	933	510	317
3h exposure, with metabolic activation
SC1	100	81	100	100	104	49	51
SC2		90			104	61	38
7.8	78	66	77	60	145	76	62
16	79	72	85	67	140	71	62
31	93	59	69	64	153	79	68
63	90	67	78	71	132	66	60
125	111	72	85	94	131	87	38
250	98	78	91	89	118	60	53
500	96	83	96	93	121	67	49
1000^(1)	76	93	108	82	111	67	38
CP	50	32	37	18	2316	1167	748

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency;

RTG = Relative Total Growth; SC = Solvent control = DMSO;

MMS = Methylmethanesulfonate; CP = Cyclophosphamide

(1) = The test item precipitated in the exposure medium

 

Table 3: Experiment 2: Cytotoxic and mutagenic response of the test substance ester in the mouse lymphoma

l5178Y test system

Dose	RSG	CE day2	RCE	RTG	Mutation frequency per 10^6survivors
(µg/mL)	(%)	(%)	(%)	(%)	total	small	large
24 h exposure, without metabolic activation
SC1	100	88	100	100	104	51	48
SC2		91			93	44	45
7.8	110	94	105	115	85	37	45
16	106	93	104	110	89	46	40
31	113	66	74	84	119	45	69
63	100	77	86	86	149	81	60
125	101	80	90	91	128	80	42
250	93	71	80	74	147	61	79
500	84	81	91	77	116	46	65
700^(1)	65	71	80	52	133	69	58
MMS	97	55	61	59	789	388	317

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency;

RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate

(1) = The test item precipitated in the exposure medium

 

Table 4: Experiment 1: Cell Counts and Subculture Data (With Metabolic Activation)

	Day 0	Day 1		Day 2
Dose	total amount of cells before treatment (x 10^6)	cell count (x10^5c/ml)	subculture x 10^6 total amount ^(1)	cell count (x10^5c/ml)	RSG	SG
(µg/mL)					(%)
SC1	8	4.8	4.0	7.9	100	19
SC2	8	4.4	4.0	8.0		18
7.8	8	3.7	4.0	7.7	78
16	8	3.5	4.0	8.2	79
31	8	4.1	4.0	8.3	93
63	8	4.1	4.0	8.0	90
125	8	5.4	4.0	7.5	111
250	8	4.4	4.0	8.1	98
500	8	4.7	4.0	7.5	96
1000^(2)	8	3.6	4.0	7.7	76
CP	8	2.5	4.0	7.2	50

Note: all calculations were made without rounding off

(1) = cell density 1.25 x 10⁵c/ml

(2) = the test item precipitated in the exposure medium

 

Table 5: Experiment 1: Selection data and cloning efficiency

Dose (µg/mL)	Mutant colonies													Cloning efficiency (at day 2)				Mutation frequency per 10^6survivors
	number of wells with mutants per selection plate^(1)										Total number of mutants			No. of empty wells per cloning plate		Total number of empty wells	CE x 100%
	1		2		3		4		5					1	2			total	small	large
without metabolic activation
	s	1	s	1	s	1	s	1	s	1	s	1	s + 1
SC1	3	7	10	6	3	6	11	4	11	4	38	27	65	47	46	93	72	100	57	40
SC2	6	6	6	16	9	6	8	10	9	11	38	49	87	47	49	96	69	144	59	78
31	4	3	4	7	7	6	3	5	6	6	24	27	51	43	39	82	85	66	30	34
63	3	6	5	4	6	6	4	5	6	5	24	26	50	63	51	114	52	106	49	53
125	7	6	14	6	13	8	9	11	11	6	54	37	91	54	36	90	76	139	79	53
250	8	5	10	6	10	10	9	5	4	8	41	34	75	41	34	75	94	90	47	39
500	8	13	7	8	7	6	12	7	7	14	41	48	89	41	45	86	80	128	56	66
600	10	6	13	10	11	10	8	9	15	7	57	42	99	36	39	75	94	123	67	49
700 (2)	12	9	14	9	7	4	8	6	14	6	55	34	89	42	48	90	76	135	80	48
800 (2)	12	8	6	8	7	7	12	9	12	7	49	39	88	45	38	83	84	121	64	51
MMS	22 26	12 16	31 23	12 11	26 18	15 15	22 15	17 17	19 19	16 13	221	144	365	56	59	115	51	933	510	317
with metabolic activation
	s	1	s	1	s	1	s	1	s	1	s	1	s + 1
SC1	8	8	7	7	7	9	9	7	6	7	37	38	75	42	43	85	81	104	49	51
SC2	8	7	10	7	11	5	14	3	7	10	50	32	82	39	39	78	90	104	61	38
7.8	10	7	13	5	12	8	7	8	4	10	46	38	84	48	51	99	66	145	76	62
16	9	8	6	8	13	7	11	8	8	10	47	41	88	44	49	93	72	140	71	62
31	7	5	7	13	11	5	5	7	13	7	43	37	80	55	51	106	59	153	79	68
63	8	11	10	10	7	5	7	5	9	6	41	37	78	50	48	98	67	132	66	60
125	22	3	10	6	8	7	8	4	9	6	57	26	83	49	44	93	72	131	87	38
250	6	11	9	4	9	8	13	9	6	6	43	38	81	45	43	88	78	118	60	53
500	11	7	10	8	11	8	12	9	6	5	50	37	87	38	46	84	83	121	67	49
1000^(2)	10	10	10	7	7	6	15	3	14	7	56	33	89	39	37	76	93	111	67	38
CP	33 28	16 20	35 22	16 23	26 32	28 23	28 30	18 21	26 36	21 16	296	202	498	76	64	140	32	2316	1167	748

s = small colonies

l = large colonies

(1) = Solvent controls and treatment groups five plates with 2000 cells/well and the positive controls ten plates with 1000 cells/well

(2) = The test item precipitated in the exposure medium

MMS, Methylmethanesulfonate

CP, Cyclophosphamide

 

Table 6: Experiment 2: Cell Counts and Subculture Data (Without Metabolic Activation)

	24 h Treatment			Day 1		Day 2
Dose	total amount of cells before treatment (x 10^6)	total amount of cells after treatment (x 106)	subculture x 10^6 total amount ^(1)	cell count (x10^5c/ml)	subculture x 10^6 total amount ^(1)	cell count (x10^5c/ml)	RSG	SG
(µg/mL)							(%)
SC1	6	22.0	4.0	5.3	4.0	7.0	100	87
SC2	6	22.0	4.0	5.4	4.0	6.8	110	86
7.8	6	22.0	4.0	5.5	4.0	7.3	106
16	6	22.4	4.0	5.8	4.0	6.6	113
31	6	24.4	4.0	5.2	4.0	7.2	100
63	6	23.2	4.0	5.3	4.0	6.6	101
125	6	23.4	4.0	5.1	4.0	6.8	93
250	6	21.8	4.0	5.0	4.0	6.9	84
500	6	19.2	4.0	5.4	4.0	6.6	65
700^(2)	6	14.6	4.0	5.7	4.0	6.3	97

Note: all calculations were made without rounding off

(1) = cell density 1.25 x 10⁵c/ml

(2) = the test item precipitated in the exposure medium

 

Table 7: Experiment 2: Selection data and cloning efficiency

Dose (µg/mL)	Mutant colonies													Cloning efficiency (at day 2)				Mutation frequency per 10^6survivors
	number of wells with mutants per selection plate^(1)										Total number of mutants			No. of empty wells per cloning plate		Total number of empty wells	CE x 100%
	1		2		3		4		5					1	2			total	small	large
without metabolic activation
	s	1	s	1	s	1	s	1	s	1	s	1	s + 1
SC1	12	6	10	9	7	12	9	6	3	6	41	39	80	42	38	80	88	104	51	48
SC2	5	7	5	11	8	9	9	7	10	4	37	38	75	39	38	77	91	93	44	45
7.8	11	8	8	6	8	10	3	6	2	9	32	39	71	39	36	75	94	85	37	45
16	9	6	5	7	9	9	10	4	6	8	39	34	73	35	41	76	93	89	46	40
31	5	8	3	9	8	8	5	10	7	7	28	42	70	49	50	99	66	119	45	69
63	9	5	7	16	15	9	14	9	11	3	56	42	98	50	39	89	77	149	81	60
125	17	7	14	10	9	3	8	4	10	7	58	31	89	37	49	86	80	128	80	42
250	9	9	8	16	6	10	8	11	9	5	40	51	91	46	48	94	71	147	61	79
500	8	8	8	11	9	6	3	11	7	12	35	48	83	48	37	85	81	116	46	65
700^(2)	7	8	12	7	6	9	8	10	12	4	45	38	83	45	49	94	71	133	69	58
MMS	10 21	20 11	17 14	16 23	23 17 15	15 13	23 15	15 15	25 19	12 13	184	153	337	53	58	111	55	789	388	317

s = small colonies

l = large colonies

(1)= Solvent controls and treatment groups five plates with 2000 cells/well and the positive controls ten plates with 1000 cells/well

(2)= The test item precipitated in the exposure medium

MMS, Methylmethanesulfonate

CP, Cyclophosphamide

 

Table 8: Historical control data of the spontaneous mutation frequencies of the vehicle controls for the mouse lymphoma assay

	Mutation frequency per 106 survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	101	98	100
SD	30	31	30
n	279	262	293
Upper control limit (95% control limits)	170	162	165
Lower control limit (95% control limits)	31	34	36

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between

September 2015 and September 2018.

 

Table 9: Historical control data of the spontaneous mutation frequencies of the positive controls for the mouse lymphoma assay

	Mutation frequency per 106 survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	803	695	1545
SD	253	223	887
n	142	132	151
Upper control limit (95% control limits)	1533	1270	3954
Lower control limit (95% control limits)	72	119	-864

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between September

2015 and September 2018.

Conclusions:

Under the conditions of the conducted test the substance did not induce forward muatation inthe thymidine kinase gene in L5178Y TK+/- 3.7.2C mouse lymphoma cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Several genotoxicity tests were conducted with the registration substance to evaluate its genotoxic potential. In detail, the following studies have been performed with the test substance:

 

- Genetic toxicity in bacteria (Ames)

A bacterial gene mutation assay (Ames test) with Bis(2-(2-butoxyethoxy)ethyl) adipate was performed according to OECD Guideline 471 and in compliance with GLP (The American Chemistry Council, 2011). Two independent experiments were performed (plate incorporation and pre-incubation), both in the presence or absence of metabolic activation (S9 mix), in the S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A pKM 101 with test substance concentrations ranging from 0.15 to 5000 µg/plate for an exposure period of 48 h. No cytotoxicity was observed in any of the tester strains up to the limit concentration of 5000 µg/plate. No increase in mean revertant number was observed in any bacterial strain after exposure to the test substance at any test concentration in both experiments. The solvent and positive controls showed the expected results and thus confirmed the efficiency of the assay. Under the conditions of this assay, the test substance did not induce mutagenicity in the selected strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A pKM 101 in the absence and presence of metabolic activation, respectively. (was already in the dossier)

 

 

- Cytogenicity/micronucleus test in human peripheral lymphocytes

A GLP conformed micronucleus test was performed according to OECD guideline 487 (Emery Oleochemicals, 2019). The potential of the registration substance to induce micronuclei in cultured peripheral human lymphocytes was investigated, either in the presence or absence of a metabolic activation system (S9-mix).

 

A dose-range-finding test was performed to choice the appropriate doses for the main study. The following concentrations and conditions were investigated:

31.3, 62.5, 125, 250, 500 and 1000 μg/mL (+/- S9, 3h exposure) and 62.5, 125, 250, 500, 1000 and 2000 μg/mL (- S9, 24h exposure). Precipitation was observed at dose levels of 125 μg/ml and upwards after adding the test substance to the culture medium. However, the amount of precipitate decreased during incubation. After 3 h the precipitate was only observed at dose levels of 1000 μg/mL and upwards. Substantial toxicity determined by CBPI (at least 55 ± 5 % cytostasis) were observed at 1000 µg/mL (3h exposure and 24 h exposure -S9) and 2000 µg/mL (24 h exposure -S9). Doses selected for the main experiments were based on precipitation.

 

The possible clastogenicity and aneugenicity of the test substance was tested in two independent experiments.In the first cytogenetic assay lymphocytes were cultured for 46 ± 2 h and thereafter exposed to the test substance at50, 500, 750 and 1000 μg/mL,dissolved in dimethylsulfoxid,for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium and were re-suspended in 5 mL culture medium with cytochalasin B (5 μg/mL) and incubated for another 24 h. In the second cytogenetic assay lymphocytes were exposed for an extended exposure time of 24 h. Lymphocytes were cultured for 46 ± 2 h and thereafter exposed in duplicate to the test substance at50, 500, 750 and 1000 μg/mLwith cytochalasin B (5 μg/mL) for 24 h in the absence of S9-mix.

In both experiments, dimethylsulfoxid (1.0% (v/v) in the final culture medium) served as vehicle control. Mitomycin C (0.25 and 0.38 μg/mL) was used as positive control for a direct acting clastogen and colchicine (0.1 μg/mL) was used as positive control for a direct acting aneugen during the 3 and 24 h exposure period. Cyclophosphamide (15 and 17.5 μg/mL) was used as an indirect acting clastogen, requiring metabolic activation, during the 3 h exposure period.

 

In the first cytogenetic experiments, thepositive control did not give a proper response in the presence of S9-mix and was thus rejected. The first experiment (1A) was therefore repeated. Since precipitates were observed at 750 and 1000 mg/mL in the first experiment, the following concentrations were used in the repeat experiment (1A) 50, 500 and 750 μg/mL.

 

In both, the first (1A) and the second cytogenicity experiment, the test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix.

 

In the first cytogenetic assay, 1 A, precipitate was observed at the highest dose level (750 μg/ml). Substantial toxicity (at least 55 ± 5 % cytostasis) was not determined at any test concentration.

In the second cytogenetic assay, precipitate was observed at the two highest dose levels (750 and 1000 μg/ml). In addition, substantial toxicity was reached at 750 μg/ml. Therefore concentrations of 50, 500 and 750 μg/ml were selected for scoring of micronuclei.

 

Vehicle and positive controls proofed the validity of the study. The number of mono- and binucleated cells with micronuclei found in the vehicle control was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. Colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

In conclusion, the test substance was not clastogenic or aneugenic in human lymphocytes under the experimental conditions.

 

 

- Gene mutation in mammalian cells

A GLP guideline study on gene mutation in mammalian cells was performed according to OECD Guideline 490 (Emery Oleochemicals, 2019). In this study the ability of the test material to induce forward mutations at the thymidine kinase (TK) locus was investigated in L5178Y mouse lymphoma cells, either in the absence (3h or 24 h exposure period) or presence (3h exposure period) of a metabolic system (S9-mix), prepared from male rats treated with phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

 

Preliminary testing of test substance solubility in dimethyl sulfoxide revealed precipitates in the exposure medium at concentrations of 1000 μg/mL and above. Therefore, 1000 µg/mL was used as the highest test substance concentration in the dose range-finding test.

 

In this dose-range finding test, lymphoma cells were treated with the test item, dissolved in dimethyl sulfoxide at 63, 125, 250, 500 and 1000 μg/mL (3 h, +/- S9 and 24 h, -S9). Both for the dose range finding experiment and the main tests, dimethyl sulfoxide at a concentration of 1% (v/v) served as negative control. Cyclophosphamide, dissolved dissolved in Hanks’ balanced salt solution (HBSS) without calcium and magnesium, was used as positive control for an indirect acting mutagen, requiring metabolic activation at an concentration of 7.5 μg/mL (3 h exposure period). Methyl methanesulfonate, dissolved dimethyl sulfoxid, was used as a direct acting mutagen at concentrations of 15 and 5 μg/mL (3 and 24 h exposure periods, respectively).

 

Precipitates were observed at the highest test substance concentration at all treatment conditions. Cytotoxicity, determined by relative suspension growth was 74% at 500 μg/mL and 0% at 1000 µg/mL compared to the relative suspension growth of the solvent control in the absence of metabolic activation (3h exposure). With metabolic activation, no toxicity determined as relative suspension growth was observed up to the highest test substance concentration of 1000 μg/mL compared to the vehicle control (3h exposure). The relative suspension growth was 37% at an test substance concentration of 1000 μg/mL compared to the relative suspension growth of the vehicle control without metabolic activation. (24h exposure).

Based on precipitation and cytotoxicity in the dose-range finding test the following concentrations were used in the first mutagenicity test (3 h exposure): 16, 31, 63, 125, 250, 500, 600, 700, 800, 900 and 1000 μg/mL (- S9) and 7.8, 16, 31, 63,125, 250, 500 and 1000 μg/mL (+ S9). The second mutagenicity test (24 h exposure) was conducted with the following test substance concentrations: 7.8, 16, 31, 63, 125, 250, 500 and 700 μg/mL (- S9).

 

In the first experiment, the test item precipitated in the culture medium at dose levels of 700 μg/mL and above.

Relative total growth (RTG) was reduced to 20% in the absence of S9-mix at a dose of 800 µg/ml and above and doses between 31 and 800 µg/mL were selected for evaluation. No substantial toxicity was observed in the presence of S9-mix, thus all dose levels were evaluated. Both in the presence and absence of S9-mix, no biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test substance. The numbers of small and large colonies were comparable to the numbers of small and large colonies of the vehicle controls.

In the second experiment, no severe toxicity was observed up to and including the highest tested dose level and all dose levels were evaluated. Precipitates occurred at the highest dose level tested. Also after a longer exposure period, no biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test substance without metabolic activation. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

 

Vehicle and positive controls proved the validity of the study. The mutation frequency of the vehicle control was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. The positive control substances, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequencies of the positive controls were within the 95% control limits of the distribution of the historical positive control database.

 

In conclusion, both in the first (3h exposure) and second (24 h exposure) mutation test the test substance did not induce a biologically relevant increase in the mutation frequency in the absence and presence of metabolic activation. Therefore, the test substance was not mutagenic in the mouse lymphoma L5178Y test system under the described conditions.

 

Conclusion on genetic toxicity

Overall, the available in vitro studies on genetic toxicity do not indicate that the test substance exhibits genotoxic properties. The test substance did not induce gene mutations in bacteria or mammalian cells and did not induce micronuclei in peripheral human lymphocytes.

Justification for classification or non-classification

The available genotoxicity data obtained for the test substance are conclusive but not sufficient for classification according to Regulation (EC) No. 1272/2008 (CLP).