Fatty acids, C16-18 (even numbered, C18 unsaturated), 2-ethylhexyl esters, epoxidized

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The endpoint mutagenicity is covered with an AMES test (OECD 471, GLP, 2021) and a MNT in vitro study (OECD 487, GLP, 2021). The test substance has been found to be negative in these two assays. Furthermore, two HPRT tests with structurally similar substances are available. Both structurally similar substances were negative in the respective assays.

Taking all these results into consideration, no indications exist for a mutagenic potential of the test substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2021

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:

26 Jun 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0022865630
- Test substance No.: 12/0076-2
- Date of production: 27 May 2020
- Expiry date: 27 May 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity: The homogeneity of the test substance was guaranteed on
account of the high purity and was ensured by mixing before preparation of the test substance solutions.
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed until 27 May 2021 as indicated by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
- Stability of the test substance in the solvent/vehicle: The stability of the test substance in the vehicle acetone was not determined analytically, because the test substance was administered immediately after preparation and is usually
stable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in acetone. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before use.

FORM AS APPLIED IN THE TEST (if different from that of starting material):
- dissolved in the vehicle acetone

OTHER
- Physical state, appearance: liquid, yellowish, clear

Target gene:

his, trp

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: BASF SE; in an AAALAC approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive
- method of preparation of S9 mix: At least 5 male Wistar rats [Crl:WI(Han)] received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. 24 hours after the last administration, the rats were sacrificed, and the livers were weighed, washed and homogenized. After centrifugation of the homogenate at 9000 x g, portions of the supernatant (S9 fraction) were stored at -70°C to -80°C. The S9 mix was prepared freshly prior to each experiment (1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors))

Test concentrations with justification for top dose:

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

3rd Experiment
Strain: TA 1537
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: Due to technical reason an evaluation of the revertant colonies of the
tester strain TA1537 was not possible; therefore, the experimental
part was repeated.

As long as precipitation did not interfere with the colony scoring, 5 mg/plate was
generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: with S9 mix: 2-aminoanthracene (2-AA); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD)

Remarks:

Sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at testing: approx. 10^9 cells per mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours

DETERMINATION OF CYTOTOXICITY
- Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Evaluation criteria:

Acceptance criteria
The experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and
TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 1535

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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

Positive controls validity:

valid

Additional information on results:

TOXICITY
- A weak bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at 5000 μg/plate.
In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions at and above 2500 μg/plate. (see table 1)

HISTORICAL CONTROL DATA
- Positive historical control data: see table 2
- Negative (solvent/vehicle) historical control data: see table 3

Table 1: Toxicity: Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	Without	-
	With	5000	-	-	5000	-
2nd-PIT	Without	5000	-	-	-	-
	With	-
3rd-PIT	Without	not tested	not tested	5000	not tested	not tested
	With	not tested	not tested	2500 - 5000	not tested	not tested

Table 2: Historical Positive Controls

Strain	S9 Mix	Positive control	No. of plates	No. of values	Min	Max	Mean	SD
TA 1535                    Without		MNNG	200	73	406	8303	4032	1720.0
With		2-AA	207	73	99	433	235	100.3
TA 100                   Without		MNNG	201	73	611	6430	2853	1237.9
With		2-AA	204	73	534	4037	2080	965.0
TA 1537                 Without		AAC	204	73	342	2381	1008	364.6
With		2-AA	210	73	46	301	161	66.7
TA 98                    Without		NOPD	198	73	391	1711	693	204.0
With		2-AA	201	73	337	3025	1676	754.9
E. coli                   Without		4-NQO	195	73	344	2265	987	423.9
With		2-AA	198	72	98	276	206	36.2

Table 3: Historical Negative Controls (based on Sorcerer Counter / Ames Study Mananger System (Dec 2019 - Dec 2020))

Strain	S9 Mix	Vehicle	No. of plates		No. of values		Min	Max	Mean	SD
TA 1535                    Without		(Alle)	288		107		6	20	13	2.8
With		(Alle)	297		107		7	21	13	2.7
TA 100                      Without		(Alle)	288		107		73	157	112	13.5
With		(Alle)	300		107		78	145	113	14.0
TA 1537                    Without		(Alle)	288		107		4	16	10	2.1
With		(Alle)	294		107		5	14	10	2.0
TA 98                        Without		(Alle)	282		107		13	31	22	3.2
With		(Alle)	294		107		15	53	26	5.4
E. coli                        Without		(Alle)	285		107		18	38	27	4.3
With		(Alle)	288		107		19	39	27	3.9

 

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT); 33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: Precipitation of the test substance was observed at and above 1000 μg/plate with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 2500 μg/plate.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 2020 - Aug 2021

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0022865630
- Test substance No.: 12/0076-2
- Date of production: 27 May 2020
- Expiry date: 27 May 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
- Storage condition of test material: ambient (RT)
- Storage Stability: The stability of the test substance under storage was guaranteed until 27 May 2022 as indicated by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
- Stability of the test substance in the solvent/vehicle: The stability of the test substance at room temperature in the vehicle acetone over a period of 4 hours was verified analytically. The analyses were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany (BASF-Project No.:01Y0076/12L029).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The substance was dissolved in acetone. The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from according to the planned doses. All test substance solutions were prepared immediately before administration.

FORM AS APPLIED IN THE TEST (if different from that of starting material):
- dissolved in the vehicle acetone

OTHER
- Physical state, appearance: liquid, yellowish, clear

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood samples from healthy non-smoking donors not receiving medication; fresh blood was collected from a single donor for each experiment
- Suitability of cells: The lymphocytes of each donor have previously shown to respond well to stimulation of proliferation with phytohemagglutinin (PHA) and to the used positive control substances.
- Sex, age and number of blood donors: Experiment I: one female donor (24 years old); Experiment II: one male donor (29 years old)
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
• Culture medium: DMEM / Ham's F12 (1:1) (DMEM/F12) medium containing stable glutamine supplemented with 10% [v/v] fetal calf serum (FCS).
• Treatment medium (4 hours treatment without and with S9 mix): DMEM/F12 medium containing stable glutamine
• Treatment medium (20 hours treatment without S9 mix): DMEM/F12 medium containing stable glutamine and supplemented with 10% [v/v] FCS.

All media were supplemented with:
• 1% [v/v] penicillin/streptomycin (final concentration 100 μg/mL)
• 1% [v/v] HEPES buffer (1 M)
For the stimulation the medium was supplemented with:
• 0.5% [v/v] Phytohemagglutinin (PHA, stock solution 0.6 mg/mL, final conc.
3 μg/mL) in the 1st Experiment and 1.5% PHA M form in the 2nd Experiment
• 0.5% [v/v] sodium heparin (25000 IE)

For the Cytochalasin B (Cyt B) treatment the medium was supplemented with:
• 30 μL Cyt B (Cyt B, stock solution: 2 mg/mL in DMSO, final conc.: 6 μg/mL)

Cytokinesis block (if used):

The commonly applied cytokinesis block method using the actin polymerisation inhibitor cytochalasin B (Cyt B) was used to discriminate between proliferating and non-proliferating cells.
- 30 μL Cyt B (Cyt B, stock solution: 2 mg/mL in DMSO, final conc: 6 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: BASF SE; in an AAALAC approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive
- method of preparation of S9 mix: At least 5 male Wistar rats [Crl:WI(Han)] received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. 24 hours after the last administration, the rats were sacrificed, and the induced livers were weighed, washed and homogenized. After centrifugation of the homogenate at 9000 x g, portions of the supernatant (S9 fraction) were stored at -70°C to -80°C. The S9 mix was prepared freshly prior to each experiment (1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors))

Test concentrations with justification for top dose:

Following the requirements of the current OECD Guideline 487 a test substance with defined composition should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. When the test substance is not of defined composition, e.g. substance of unknown or variable composition, complex reaction products or biological materials (socalled UVCBs), or environmental extracts, the top concentration should be higher to increase
the concentration of each of the components (e.g. 5 mg/mL). In case of toxicity, the top concentration should produce reduction of the proliferation index (CBPI) to 45 ± 5% (leading to a cytostasis increase of 55 ± 5%) of the concurrent vehicle control. For relatively insoluble test substances only one concentration should be tested showing turbidity or precipitation in culture medium at the end of exposure period.

In this study the concentrations are given as rounded values by using a dilution factor of 1.8, in general. At least three concentrations were evaluated to detect a possible dose-response relationship. At least 2 cultures were prepared per test group (referred to as A and B), and at least 1000 cells per culture were evaluated for the occurrence of micronucleated cells.

Vehicle / solvent:

- vehicle: acetone
- Justification for choice of vehicle: Due to the insolubility of the test substance in water, acetone was selected as the vehicle, which has been demonstrated to be suitable in the in vitro micronucleus test and for which historical control data are available.
- The final concentration of the vehicle acetone in culture medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control cultures with and without S9 mix contained the vehicle selected for the test substance in the same volume and concentration as used in the test cultures.

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE: :
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Stimulating time: 48h (1:10 blood : culture medium with PHA + sodium heparin)
- Exposure duration: 4h (Exp. I); 20 h (Exp. II)
- Harvest time after the end of treatment (Recovery time): 16h (Exp. I)
- Harvest time: 20h

FOR MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Cytochalasin B (30 μL Cytochalasin B (Cyt B, stock solution: 2 mg/mL in DMSO, final concentration: 6 μg/mL)


METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cells were centrifugated (900 g, 5 min) and washed with HBSS
- cells were centrifuged (900 g, 5 min) and suspended in 0.0375 M KCl (37°C)
- incubation for 20 min at 37°C
- cells were fixed by adding of fixative (19 parts methanol and 1 part acetic acid)
- cells were centrifuged (900 g, 5 min, 4°C) and suspended in fresh fixative
- incubation for 20 min at 4°C
- fixation step will be repeated twice
- cells can be centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides
- slides were dipped in deionized water
- cells were pipetted on the slide and fixed by passing through a flame

- cells were stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2,10 min) and mounted

NUMBER OF CELLS EVALUATED:
- at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).
- The diameter of the micronucleus was less than 1/3 of the main nucleus
- The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
- Only binucleated cells were scored.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: determination of CBPI (Cytokinesis-block proliferation index) in 500 cells per culture (1000 cells per test group); A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis. CBPI = ((Mononucleate cells x 1) + (Binucleate cells x 2) + (Multinucleate cells x 3)) / Total number of cells

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

An appropriate statistical analysis was performed. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE). If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (s) were printed in the tables. In addition, a statistical trend test (SAS procedure REG) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the number of micronucleated cells and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05. However, both, biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH values were not relevantly influenced by test substance treatment
- Data on osmolality: Osmolality was not relevantly influenced by test substance treatment
- Precipitation and time of the determination::
In the absence of S9 mix, test substance precipitation was observed macroscopically and microscopically in culture medium at the end of treatment at 18.5 μg/mL and above in the 1st Experiment and macroscopically at 18.5 μg/mL in the 2nd Experiment.
In the presence of S9 mix precipitation was observed macroscopically and microscopically at the highest dose 33.3 μg/mL in the 1st Experiment

CYTOTOXICITY
- In this study, no reduced proliferative activity was observed either after 4 hours exposure interval in the absence and presence of S9 mix or after 20 hours continuous test substance treatment without S9 mix in the test groups scored for cytogenetic damage.

STUDY RESULTS
1st Experiment:
- values (0.3 – 0.5% micronucleated cells) were within the range of the 95% control limit of the historical negative control data range
- No statistical significance compared to the vehicle control value

2nd Experiment:
- values (0.3 – 0.4% micronucleated cells) were within the range of the 95% control limit of the historical negative control data range
- No statistical significance compared to the vehicle control value

None of the data in the two experimens showed a positive dose response as assessed by a trend analysis.

- positive control substances MMC, Colchicine and CPA induced micronucleus frequencies in all independently performed experiments (compatible to the historical positive control data range)

HISTORICAL CONTROL DATA
- Positive historical control data: see tables 6 and 7
- Negative (solvent/vehicle) historical control data: see tables 8 and 9

Table 4: Summary table - experimental parts without S9 mix

Exp.	Exposure/ Recovery/ Preparation interval	Test groups [µg/mL]	S9 mix	Prec.*	Micro- nucleated cells** [%]	Cytotoxicity Proliferation index cytostasis [%]
1	4/16/20	Vehicle control (*1)	-	n.d.	0.5	0
		1	-	-	n.d.	2.9
		1.8	-	-	n.d.	-0.2
		3.2	-	-	n.d.	-2
		5.7	-	-	0.3	3.4
		10.3	-	-	0.4	4.4
		18.5	-	+	0.5	0.7
		33.3	-	+	n.p.	n.p.
		60	-	+	n.p.	n.p.
		Positive control (*2)	-	n.d.	7.5 (S)	20
2	20/0/20	Vehicle control (*1)	-	n.d.	0.5	0
		1	-	-	n.d.	14.9
		1.8	-	-	n.d.	14.4
		3.2	-	-	n.d.	16.2
		5.7	-	-	0.3	13.7
		10.3	-	-	0.3	10
		18.5	-	+	0.4	3
		33.3	-	+	n.p.	n.p.
		60	-	+	n.p.	n.p.
		Positive control (*3)	-	n.d.	4.4 (S)	10.4
		Positive control (*4)	-	n.d.	6.7 (S)	23.2

* Precipitation in culture medium at the end of exposure period (microscopically and/or macroscopically)
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
(S) Frequency statistically significantly higher than corresponding control values
n.d. Not determined
n.p. Not continued due to strong test substance precipitation
(*1) Aceton 1% (v/v) (*2) MMC 0.31 μg/mL
(*3) MMC 0.04 μg/mL (*4) Col 0.05 μg/mL

Table 5: Summary table - experimental part with S9 mix

Exp.	Exposure/ Recovery/ Preparation interval	Test groups [µg/mL]	S9 mix	Prec.*	Micro- nucleated cells** [%]	Cytotoxicity Proliferation index cytostasis [%]
1	4/16/20	Vehicle control (*1)	+	n.d.	0.4	0
		0.5	+	-	n.d.	3.8
		1	+	-	n.d.	5.2
		1.8	+	-	n.d.	6.4
		3.2	+	-	n.d.	9
		5.7	+	-	n.d.	7.1
		10.3	+	-	0.4	14
		18.5	+	-	0.4	3.5
		33.3	+	+	0.5	8.4
		Positive control (*5)	+	n.d.	2.5 (S)	12.1
		Positive control (*6)	+	n.d.	2.9 (S)	17.7

* Precipitation in culture medium at the end of exposure period (microscopically and/or macroscopically)
** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group
(S) Frequency statistically significantly higher than corresponding control values
n.d. Not determined
n.p. Not continued due to strong test substance precipitation
(*1) Aceton 1% (v/v) (*5) CPA 2.50 μg/mL (*6) CPA 3.50 μg/mL

Table 6: HISTORICAL POSITIVE CONTROL DATA; WITHOUT S9 MIX, Mitomycin C and Colchicin; Period: April 2018 – November 2020

Micronucleated cells [%]
Exposure period	4 hrs	20 hrs	20 hrs
Substance and concentration	MMC 0.31 µg/mL	MMC 0.04 µg/mL	Col 0.05 µg/mL
Mean	9.1	4.1	4.0
Minimum	3.8	2.1	2.4
Maximum	15.1	7.1	7.2
Standard Deviation	2.99	0.93	1.05
No. of Experiments	56	48	45

Table 7: HISTORICAL POSITIVE CONTROL DATA; WITH S9 MIX, Cychlophosphamid; Period: April 2018 – November 2020

Micronucleated cells [%]
Exposure period	4 hrs
Substance and concentration	CPA 2.50 µg/mL
Mean	3.6
Minimum	1.9
Maximum	6.9
Standard Deviation	1.12
No. of Experiments	55

Table 8: HISTORICAL NEGATIVE CONTROL DATA; WITHOUT S9 MIX, ALL VEHICLES; Period: April 2018 – November 2020

Micronucleated cells [%]
Exposure period	4 hrs	20 hrs
Mean	0.5	0.5
Minimum	0.1	0.2
Maximum	1.0	1.2
Standard Deviation	0.18	0.17
95% Lower Control Limit	0.2	0.2
95% Upper Control Limit	0.9	0.9
No. of Experiments	59	54

Table 9: HISTORICAL NEGATIVE CONTROL DATA; WITH S9 MIX, ALL VEHICLES; Period: April 2018 – November 2020

Micronucleated cells [%]
Exposure period	4 hrs
Mean	0.6
Minimum	0.3
Maximum	1.3
Standard Deviation	0.24
95% Lower Control Limit	0.1
95% Upper Control Limit	1.1
No. of Experiments	59

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that the test substance has no potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and b-naphthoflavone induced rats (exogenous metabolic activation).
According to an initial range-finding solubility test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated for the occurrence of micronuclei:

1st Experiment

4 hours exposure, without S9 mix
0; 1.0; 1.8; 3.2; 5.7; 10.3; 18.5; 33.3; 60.0 μg/mL
4 hours exposure, with S9 mix
0; 0.5; 1.0; 1.8; 3.2; 5.7; 10.3; 18.5; 33.3 μg/mL

2nd Experiment
20 hours exposure, without S9 mix
0; 1.0; 1.8; 3.2; 5.7; 10.3; 18.5; 33.3; 60.0 μg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.
In this study, acetone was selected as vehicle. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicin and Cyclophosphamide (CPA), led to the expected increase in the number of cells containing micronuclei.
In this study, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration.
On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei  either without S9 mix or after adding a metabolizing system.
Thus, under the experimental conditions described, the test substance is considered to have no chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

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

Specific details on test material used for the study:

- Test-substance No.: 12/0029-1
- Lot/batch No.: CE80580015
- Expiration Date: February 27, 2013
- Name of test material (as cited in study report): Sovermol 1055

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

see any other information on materials and methods incl. tables

Vehicle / solvent:

On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol. The final concentration of ethanol in culture medium was 0.5% v/v.

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

DURATION
- Preincubation period: 24 hours
- After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10% FBS, in the absence of metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5E6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.

NUMBER OF REPLICATIONS:
- The study was performed in two independent experiments, using identical experimental procedures.

NUMBER OF CELLS EVALUATED:
- The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
- The numbers of mutant colonies per 1E6 cells found in the solvent controls falls within the laboratory historical control data.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The cloning efficiency II (absolute value) of the solvent controls should exceed 50%.
- The data of this study comply with the above mentioned criteria.

Evaluation of Results:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
- A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
- A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

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

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 examined

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Phase separation was observed in experiment I at 120 μg/mL with and without metabolic activation. In experiment II phase separation occurred at 120.0 μg/mL with metabolic activation.

RESULTS GENOTOXICITY:
No relevant and reproducible increase in mutant colony numbers/1E6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency exceeded the threshold of three times the mutation frequency of the solvent control in the second experiment without metabolic activation at 60.0 μg/mL in culture I and at 7.5 to 60.0 μg/mL in culture II. However, only the increase observed in culture I at 60.0 μg/mL exceeded the historical range of solvent controls (2.6 - 40.3 mutant colonies/10E6 cells). The apparent increase of the mutation frequency in culture II is based on the low solvent control of just 3.9 colonies per 1E6 cells. The absolute values of the mutation frequency remained within the historical range of solvent controls. Furthermore, the apparent increase in culture II was not dose dependent as indicated by the lacking statistical significance. Consequently, the isolated increase noted in the first culture at 60.0 μg/mL was judged as irreproducible fluctuation. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of experiment I without metabolic activation and in the first culture of experiment II without metabolic activation. However, the trends were judged as biologically irrelevant as they were not reproduced in the parallel cultures. Furthermore, the mutation frequency of the second culture of the first experiment without metabolic activation remained within the historical range of solvent controls. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.9 up to 19.6 mutants per 1E6 cells; the range of the groups treated with the test item was from 6.1 up to 46.2 mutants per 1E6 cells. The cloning efficiency II of the solvent control of the first culture of the second experiment with metabolic activity reached but did not exceed the lower limit of 50%. The data are valid however, as the solvent control of the parallel culture exceeded this limit. EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 4000 μg/mL limited by the solubility of the test item in ethanol and aqueous medium. Test item concentrations between 31.3 μg/mL and 4000 μg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 62.5 μg/mL and above without metabolic activation and 500.0 μg/mL and above with metabolic activation following 4 hours treatment. Following 24 hours treatment without metabolic activation cytotoxic effects as described above occurred at 62.5 μg/mL and above. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation occurred at 62.5 μg/mL and above with and without metabolic activation following 4 and 24 hours treatment. There was no relevant shift of the osmolarity and pH value even at the maximum concentration of the test item.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 120.0 μg/mL without metabolic activation and in the second experiment at 60.0 μg/mL without metabolic activation.

Conclusions:

The test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

A GLP-compliant gene mutation assay, tested according to OECD guideline 476, was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The test item was dissolved in ethanol. The concentration range of the main experiments was limited by phase separation of the test item and cytotoxic effects. Phase separation was observed in experiment I at 120 μg/mL with and without metabolic activation and in experiment II at 120.0 μg/mL with metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 120.0 μg/mL without metabolic activation and in the second experiment at 60.0μg/mL without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 15, 1992 - June 25, 1992

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)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk gene locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK +/- mouse lymphoma cells were obtained from the American Type Culture Collection. They were stored as frozen stocks in liquid nitrogen. Each batch of cells was purged of TK mutants, checked for spontaneous mutant frequency and that they were mycoplasma free. For each experiment the vials were thawed rapidly, the cells were diluted in RPMI 10 and incubated in a humidified atmosphere of 5 % v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mammalian liver post-mitochondrial fraction

Test concentrations with justification for top dose:

The concentrations were selected using a cytotoxicity range-finder. See Table 1 below.

Vehicle / solvent:

Acetone was used as a solvent. The test item was added and the culture was then vortexed for approx. 10 seconds to obtain a good emulsion. A top dose of 5000 ug/ml was achievable using an emulsion.

Negative solvent / vehicle controls:

yes

Remarks:

Comprised of solvent, acetone, diluted 100-fold in the treatment medium.

Positive controls:

yes

Remarks:

The positive controls were also made to certain concentrations please see table 2 below

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION:

Preparations of cofactor solutions with and without S-9

Quantity (ml)
With S-9 Without S-9
Glucose-6-phosphate (180 mg/ml) 1.0 -
NADP (25 mg/ml) 1.0 -
150 mM KCL 1.0 5.0
Rat Liver S-9 2.0 -

The above were used at the rate of 1.0 ml per 19 ml of cell culture containing the test chemical (to achieve the required final concentration in a total of 20 ml).


Three types of RPMI 1640 medium were prepared as follows:
Final Concentration in:
RPMI A RPMI 10 RPMI 20
Horse Serum (heat inactivated) 0 % v/v 10 % v/v 20 % v/v
Gentamycin 100 ug/ml 100 ug/ml 100 ug/ml
Fungizone 2.5 ug/ml 2.5 ug/ml 2.5 ug/ml
Pluronic 0.5 ug/ml 0.5 ug/ml -


DURATION
- Exposure duration: 9- 15 days
- Expression time (cells in growth medium): 2 days

NUMBER OF CELLS EVALUATED: 1 x E7 cells per flask

DETERMINATION OF CYTOTOXICITY
Following adjustment of the cultures to 2 x E5 cells/ml after treatment, samples from these were diluted to 8 cells/ml as seen in table 3. using a 8-channel pipette, 0.2 ml of concentration C of each culture was placed into each well of two 96-well microtitre plates (192 wells, at an average of 1.6 cells per wall). The plates were incubated at 37 C in a humidified incubator gassed with 5 % v/v CO2 in air until scorable (9-15 days). Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:

At the end of the expression period the cell densities in the selected cultures were adjusted to 1 x E4/ml. TFT (300 ug/ml) was diluted 100-fold into these suspensions to give a final concentration of 3 ug/ml. Using a 8-channel pipette, 0.2 ml of each suspension was placed into each well of four 96-well microtitre plates (384 wells at 2 x E3 cells per well). Plates were incubated until scorable (13 days) and wells containing clones were identified as above and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored for the negative and positive controls and for doses of test chemical showing a significant increase in mutant frequency over the negative control.

Statistics:

All calculations were performed on a microcomputer.

Determination of survival or viability: Poison distribution, plating efficiency, % relative survival
Determination of mutant frequency: Mutant frequency, plating efficiency,
Statistical significance of mutant frequencies.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Please see Tables 5 & 6 below

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No marked toxicity was seen after the application of the highest dose. Please see Table 4

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Please see table 4 below

Table 4: raw plate counts and % relative survival for Epoxidised Soybean Oil in the cytotoxicity range-finder.

Treatment ug/mL)	In the absence of S-9		In the presence of S-9
	Survival (1) at Day 0*	% Relative survival	Survival (1) at day 0*	% relative survival
0	18	100.0	23	100.0
78.125	21	129.2	21	84.2
156.25	16	83.8	17	59.7
312.5	18	100.0	19	71.0
625	15	76.5	15	49.9
1250	15	76.5	15	49.9
2500	16	83.8	18	65.2
5000	19	109.0	23	100.0

(1) 1.6 cells/well plated

* 32 wells scored

Table 5: Summary of Results Experiment 1

Treatment (ug/ml)	Absence of S-9		Treatment (ug/mL)	Presence of S-9
	% RS	Mutant Frequency		% RS	Mutant Frequency
0	100.0	312.57	0	100.0	401.83
312.5	95.8	389.82 NS	312.5	97.9	484.86 NS
625	86.6	326.20 NS	625	87.9	563.19 NS
1250	79.9	439.82 NS	1250	86.0	518.57 NS
2500	131.9	520.41 *	2500	152.9	426.40 NS
5000	143.9	467.96 *	5000	178.7	433.74 NS
Linear Trend		**	Linear Trend		NS
NQO			BP
0.05	83.5	1676.58	2	55.2	1794.55
0.1	45.3	2078.73	3	39.4	2122.84

 

Table 6: Summary of Results Experiment 2

Treatment (ug/ml)	Absence of S-9		Treatment (ug/mL)	Presence of S-9
	% RS	Mutant Frequency		% RS	Mutant Frequency
0	100.0	410.98	0	100.0	304.98
312.5	112.9	334.82 NS	312.5	91.7	331.46 NS
625	87.7	466.23 NS	625	82.6	331.03 NS
1250	71.4	404.74 NS	1250	87.7	313.73 NS
2500	84.5	461.15 NS	2500	84.5	283.11 NS
5000 $$	100.7		5000	83.2	311.46 NS
Linear Trend		NS	Linear Trend		NS
NQO			BP
0.05	67.5	1136.27	2	59.5	883.78
0.1	37.7	1143.11	3	32.1	1443.04

 

NS Not significant

$$ Treatment excluded due to excessive heterogeneity

*, **, *** Significant at 5 %, 1% and 0.1 % level respectively

Conclusions:

It is concluded that, under the conditions employed in this study, the test substance failed to demonstrate the ability to induce mutation at the tk locus of L5178Y mouse lymphoma cells in the absence and presence of S-9. Therefore, the test substance is not considered to be mutagenic.

Executive summary:

The test substance was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finder followed by 2 independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).

Following a wide range of treatments in the range-finder experiment, separated by 2-fold intervals and ranging from 78.125 to 5000µg/ml, cells survived all doses of the test substance yielding 109.0 % relative survival in the absence and 100.0 % relative survival in the presence of S-9 at the top dose.

Accordingly, 5 doses were chosen for the first experiment, separated by 2-fold intervals and ranging from 312.5 to 5000µg/ml. All doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top doses plated yielded 143.9 % and 178.7 % relative survival in the absence and presence of S-9. In the second experiment the same dose range was selected. The top dose plated in this experiment was again 5000µg/ml in the absence and presence of S-9, which yielded 100.7 % and 83.2 % relative survival respectively.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In the absence of S-9, reproducible statistically significant and dose-related increases in mutant frequency were not observed in the 2 experiments over the dose range 312.5 to 2500µg/ml. At 5000µg/ml, a positive point was obtained in Experiment 1 and due to heterogeneity in the data this dose was excluded from analysis in Experiment 2. However, if each of the replicate cultures at 5000µg/ml in Experiment 2 are considered in turn, neither yields a statistically significant increase in mutant frequency. This, combined with the fact that there were no absolute increases in mutant numbers in Experiment 1 at 5000µg/ml and that carry over of the test compound was a problem at this dose, suggests that the increased mutant frequency seen in experiment 1 was not the result of chemically induced mutation.

In the presence of S-9, no statistically significant increases in mutant frequency were observed at any dose level tested in Experiment 1 or 2.

It is concluded that, under the conditions employed in this study, the test substance failed to demonstrate the ability to induce mutation at the tk locus of L5178Y mouse lymphoma cells in the absence and presence of S-9. Therefore, The test substance is not considered to be mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An Ames test as well as an in vitro MNT study are available for the test substance itself. Furthermore, two HPRT in vitro studies were performed with structurally similar substances. Summaries of these four studies are provided below.

In vitro gene mutation study in bacteria (Ames):

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT); 33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: Precipitation of the test substance was observed at and above 1000 μg/plate with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 2500 μg/plate.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF SE, 2021).

In vitro micronucleus study (MNT):

The test substance was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and b-naphthoflavone induced rats (exogenous metabolic activation).
According to an initial range-finding solubility test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated for the occurrence of micronuclei:

1st Experiment

4 hours exposure, without S9 mix
0; 1.0; 1.8; 3.2; 5.7; 10.3; 18.5; 33.3; 60.0 μg/mL
4 hours exposure, with S9 mix
0; 0.5; 1.0; 1.8; 3.2; 5.7; 10.3; 18.5; 33.3 μg/mL

2nd Experiment
20 hours exposure, without S9 mix
0; 1.0; 1.8; 3.2; 5.7; 10.3; 18.5; 33.3; 60.0 μg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.
In this study, acetone was selected as vehicle. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicin and Cyclophosphamide (CPA), led to the expected increase in the number of cells containing micronuclei.
In this study, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration.
On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei  either without S9 mix or after adding a metabolizing system.
Thus, under the experimental conditions described, the test substance is considered to have no chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation (BASF SE, 2021).

Gene mutation assay in mammalian cells (HPRT) (CAS 158318-67-3):

A GLP-compliant gene mutation assay, tested according to OECD guideline 476, was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The test item was dissolved in ethanol. The concentration range of the main experiments was limited by phase separation of the test item and cytotoxic effects. Phase separation was observed in experiment I at 120 μg/mL with and without metabolic activation and in experiment II at 120.0 μg/mL with metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 120.0 μg/mL without metabolic activation and in the second experiment at 60.0μg/mL without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Therefore, the test item is considered to be non-mutagenic in this HPRT assay (Harlan 2013).

 

Gene mutation assay in mammalian cells (HPRT) (CAS 8013-07-8):

The test substance was assayed for its ability to induce mutation at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finder followed by 2 independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).

Following a wide range of treatments in the range-finder experiment, separated by 2-fold intervals and ranging from 78.125 to 5000µg/ml, cells survived all doses of the test substance yielding 109.0 % relative survival in the absence and 100.0 % relative survival in the presence of S-9 at the top dose.

Accordingly, 5 doses were chosen for the first experiment, separated by 2-fold intervals and ranging from 312.5 to 5000µg/ml. All doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top doses plated yielded 143.9 % and 178.7 % relative survival in the absence and presence of S-9. In the second experiment the same dose range was selected. The top dose plated in this experiment was again 5000µg/ml in the absence and presence of S-9, which yielded 100.7 % and 83.2 % relative survival respectively.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In the absence of S-9, reproducible statistically significant and dose-related increases in mutant frequency were not observed in the 2 experiments over the dose range 312.5 to 2500µg/ml. At 5000µg/ml, a positive point was obtained in Experiment 1 and due to heterogeneity in the data this dose was excluded from analysis in Experiment 2. However, if each of the replicate cultures at 5000µg/ml in Experiment 2 are considered in turn, neither yields a statistically significant increase in mutant frequency. This, combined with the fact that there were no absolute increases in mutant numbers in Experiment 1 at 5000µg/ml and that carry over of the test compound was a problem at this dose, suggests that the increased mutant frequency seen in experiment 1 was not the result of chemically induced mutation.

In the presence of S-9, no statistically significant increases in mutant frequency were observed at any dose level tested in Experiment 1 or 2. Therefore, the test item is considered to be non-mutagenic in this HPRT assay (ETTEC 1992).

Justification for classification or non-classification

No indication of genotoxicity was observed in the Ames test (OECD 471, GLP, 2021) and in the MNT in vitro study (OECD 487, GLP, 2021). Furthermore, two structurally related substances were not mutagenic in the HPRT assay in vitro. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.