Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and stearic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro assays were performed to evaluate the genotoxic potential of the test substance (OECD 471, 476, 487 und GLP conditions). The test item did not induce mutations in bacterial (S. typhimurium TA 100, TA 1535, TA 1537, TA 98 and E. coli WP2) or mammalian cells (CHO cells) and did not induce micronuclei in human lymphocytes in the absence or presence of metabolic activation.

Therefore, the test item is considered as non-mutagenic under the conditions of these tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2019 - 16 January 2020

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:

yes

Remarks:

see also prinicple of test method

Principles of method if other than guideline:

A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): BASF SE, Ludwigshafen, Germany
- Lot/batch number of test material: 0020798322
- Purity, including information on contaminants, isomers, etc.: 96.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Homogeneity given. The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Due to the use of culture medium as vehicle, the verification of the stability of the test substance in the vehicle is not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Stock formulations of the test item and serial dilutions in culture medium were prepared freshly before treatment and used within two hours of preparation.
- Final concentration of a dissolved solid, stock liquid or gel: please refer to section "Test concentrations with justification for top dose".

FORM AS APPLIED IN THE TEST: dilution in culture medium

OTHER SPECIFICS
- Physical state / Appearance: solid, white
- Molecular weight: 435 g/mol

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication.
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Normal cell cycle time (negative control): not specified

For lymphocytes:
- Sex, age and number of blood donors: one male donor (20 years old) for Experiment I and one male donor (21 years old) for Experiment II.
- Whether whole blood or separated lymphocytes were used: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection.
- Whether blood from different donors were pooled or not: no
- Mitogen used for lymphocytes: yes, PHA

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Phenobarbital/ß-naphthoflavone induced rat liver S9
- source of S9: The S9 was prepared and stored according to the currently valid version of the ICCR-Roßdorf GmbH SOP for rat liver S9 preparation.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentration of the S9 preparation used for this study was 30.2 mg/mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-Aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

- Exp. I: 13.4; 23.4; 40.9; 71.6; 125; 219; 384; 671; 1175; 2056 µg/mL (with and without S9 mix)
- Exp. II: 5.7; 8.6; 12.9; 19.3; 29.0; 43.5; 65.3; 98.0; 171; 300 µg/mL (without S9 mix)

- Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. With regard to the molecular weight and the purity of the test item, 2056 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 13.4 to 2056 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 71.6 μg/mL and above in the absence of S9 mix and at 125 μg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Due to the precipitation data, 300 μg/mL were chosen as top treatment concentration for Experiment II.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (culture medium)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

other: corresponding to solvent control

Negative solvent / vehicle controls:

yes

Remarks:

solvent control (culture medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

other: corresponding to solvent control

Negative solvent / vehicle controls:

yes

Remarks:

solvent control (culture medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Demecolcine

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE: Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 hours, stimulation for proliferation by the addition of the mitogen PHA (3 µg/mL) to the culture medium.
- Exposure duration/duration of treatment: 4 hours (Exp. I); 20 hours (Exp. II)
- Harvest time after the end of treatment (sampling/recovery times): Recovery time: 16 hours (Exp. I only) and another 20 hours after addition of Cytochalasin B (Exp. I and II); Harvest time: 40 hours after start of treatment with the test item

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: Cytochalasin B (4 μg/mL), 20 hours exposure period
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): In each experimental group two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. The frequency of micronucleated cells was reported as % micronucleated cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- A preliminary cytotoxicity test (Exp. I) was performed to determine the concentrations to be used in the main experiment (Exp. II). Cytotoxicity is characterized by the percentages of reduction in the cytokinesis-block proliferation index (CBPI) in comparison to the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. 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
Cytostasis % = 100 – 100 [(CBPI(Test item) – 1) / (CBPI(Solvent control) – 1)]

Evaluation criteria:

1. Acceptability Criteria: The micronucleus assay will be considered acceptable if it meets the following criteria:
- The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval).
- The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data compared with the concurrent solvent control and produce a statistically significant increase.
- Cell proliferation criteria in the solvent control are considered to be acceptable.
- All experimental conditions were tested unless one exposure condition resulted in a clearly positive result.
- The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
- The criteria for the selection of top concentration are consistent with those described.

2. Interpretation of Results: A test item can be classified as non-clastogenic and non-aneugenic if:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).
A test item can be classified as clastogenic and aneugenic if:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)

Statistics:

Statistical significance was confirmed by the Chi square test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control. A linear regression was performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells 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. Both, biological and statistical significance were considered together.

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:

other: corresponding to solvent control

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: No relevant influence on osmolarity or pH was observed.
- Precipitation and time of the determination: In Experiment I, precipitation of the test item in the culture medium was observed at 71.6 µg/mL and above in the absence of S9 mix and at 125 µg/mL and above in the presence of S9 mix at the end of treatment. In Experiment II in the absence of S9 mix no precipitation was observed at the end of treatment.
- Definition of acceptable cells for analysis: please, refer to the section "Evaluation criteria"

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis; p-value: 0.965 in Exp. I without S9 mix; 0.838 in Exp. I with S9 mix; 0.767 in Exp. II without S9 mix

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes, Demecolcine (100 ng/mL), MMC (0.8 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI and distribution of mono-, bi- and multi-nucleated cells: yes
o In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In Experiment II in the absence of S9 mix after continuous treatment, clear cytotoxicity (cytostasis of 56.1%) was observed at the highest evaluated concentration.

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture: yes
In this study in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

HISTORICAL CONTROL DATA: please refer to Table 1 in the section "Any other information on materials and methods incl. tables"

Table 1: Summary of results

 

Exp.	Preparation interval	Test item concentration in µg/mL	Proliferation index CBPI	Cytostasis in %*	Micronucleated cells
					in %**	95% Ctrl limit
Exposure period 4 h without S9 mix
I	40 h	Solvent control^1/#	1.64		0.45	0.01 – 1.20
		Positive control^2	1.54	15.8	8.05^S
		23.4^#	1.72	n.c.	0.50
		40.9^#	1.67	n.c.	0.60
		71.6^P/#	1.50	23.0	0.43
Trend test: p-value 0.965
Exposure period 20 h without S9 mix
II	40 h	Solvent control^1	1.94		0.20	0.00 – 1.14
		Positive control^3	1.53	43.3	4.20^S
		19.3	1.84	10.9	0.35
		43.5	1.65	31.1	0.05
		98.0	1.41	56.1	0.20
Trend test: p-value 0.767
Exposure period 4 h with S9 mix
I	40 h	Solvent control^1	1.72		0.75	0.00 – 1.24
		Positive control^4	1.50	30.9	4.15^S
		40.9	1.75	n.c.	0.35
		71.6	1.70	3.2	0.90
		125^P	1.66	7.8	0.70
Trend test: p-value 0.838

 

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

P Precipitation occurred at the end of treatment

S The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1   Culture medium

2   MMC 0.8 µg/mL

3   Demecolcine 100 ng/mL

4   CPA 17.5 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

Executive summary:

The test item, suspended in culture medium, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

 

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I
Stimulation period	48 hrs	48 hrs	48 hrs
Exposure period	4 hrs	20 hrs	4 hrs
Recovery	16 hrs	-	16 hrs
Cytochalasin B exposure	20 hrs	20 hrs	20 hrs
Total culture period	88 hrs	88 hrs	88 hrs

 

In each experimental group two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2056 µg/mL of the test item) was chosen with regard to the molecular weight and the purity of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In Experiment II in the absence of S9 mix after continuous treatment, clear cytotoxicity (cytostasis of 56.1%) was observed at the highest evaluated concentration.

In this study in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 - 26 Sep 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by Landesamt für Umwelt Rheinland-Pfalz

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): not specified
- Lot/batch number of test material: 0020798322
- Purity, including information on contaminants, isomers, etc.: 96.9 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions. The stability of the test substance under storage conditions is guaranteed until 17 Oct 2020 as indicated by the sponsor.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: 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
- 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 suspended in acetone. To achieve homogeneity 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. To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before removal. All test substance formulations were prepared immediately before use.
- Final concentration of a dissolved solid, stock liquid or gel: please refer to section "Test concentrations with justification for top dose"

FORM AS APPLIED IN THE TEST: homogeneous suspension

OTHER SPECIFICS
- Physical state, appearance: solid, white

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: liver S9 mix
- source of S9: The liver S9 fraction was prepared from 5 male Wistar rats pretreated with phenobarbital and β-naphthoflavone by the sponsor, according to Ames et al. 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: The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and mixed with S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 part of S9 fraction and 9 parts of S9 supplement (consisting of 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 15 mM phosphate buffer (pH 7.4)); 0.5 mL S9 mix in 0.1 mL test solution or vehicle, 0.1 mL fresh bacterial culture and 2 mL soft agar.
- quality controls of S9: To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.

Test concentrations with justification for top dose:

- First experiment: 0; 33; 100; 333; 1000; 2600 and 5200 μg/plate (SPT with TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with and without S9 mix)
- Second experiment: 0; 1; 3.3; 10; 33; 100 and 333 µg/plate (SPT with TA 100 without S9 mix) and 0; 3.3; 10; 33; 100; 333 and 1000 µg/plate (SPT with TA 98 with S9 mix)
- Third experiment: 0; 1; 3.3; 10; 33; 100 and 333 µg/plate (PIT with all strains without S9 mix) and
0; 3.3; 10; 33; 100; 333 and 1000 µg/plate (PIT with TA strains with S9 mix) and 0; 10; 33; 100; 333; 1000 and 2600 µg/plate (PIT with E.coli with S9 mix)
- 3 test plates per dose or per control
- In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. In this study, due to the purity of the test substance 5.2 mg/plate was used as top dose in the 1st Experiment. Bacteriotoxicity was observed in the first standard plate test. Therefore, the experimental part with tester strain TA 100 without S9 mix and TA 98 with S9 mix was repeated with adjusted doses.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility 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.
- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control: 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: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitro-o-phenylenediamine (NOPD)

Remarks:

Without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control: 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:

other: 2-aminoanthracene

Remarks:

With S9 mix

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approximately 10^9 cells per mL
- SPT: Test tubes containing 2-ml portions of soft agar are kept in a water bath at about 42 - 45°C, and 0.1 mL test solution or vehicle (negative control), 0.1 mL fresh bacterial culture and 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation) are added. After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.
- PIT: 0.1 mL test solution or vehicle added in 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
- Exposure duration/duration of treatment: After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his/trp revertants) were counted.

METHODS FOR MEASUREMENT 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 and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

METHODS FOR MEASUREMENTS OF GENOTOXICITY
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

OTHER:
- Solubility: If precipitation of the test material was observed, it would be recorded. 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. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.
- Sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.

Evaluation criteria:

- Acceptance criteria:
Generally, 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 compatible with the range of the historical positive control
data or above.
• Fresh bacterial culture containing approximately 109 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.

Statistics:

No statistical analysis was conducted.

Species / strain:

E. coli WP2 uvr A

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 examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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 examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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 examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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 examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Test substance precipitation occurred at 1000 μg/plate and above in all experimental parts with S9 mix and in the 1st SPT without S9 mix.
- Definition of acceptable cells for analysis: yes, please refer to section "Evaluation criteria".

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. The number of revertant colonies in the negative controls, with and without S9 mix, were within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies compatible with the range of the historical positive control data.
- Sterility control data: first experiment: no growth at 5200 µg test substance, 0.5 mL vehicle, 0.5 mL buffer, 0.5 mL S9 mix, 2.0 mL soft agar; second and third experiment: no growth at 2600 µg test substance, 0.5 mL vehicle, 0.5 mL buffer, 0.5 mL S9 mix, 2.0 mL soft agar

Ames test:
- Signs of toxicity: A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was observed in the SPT without S9 mix at and above 333 μg/plate (TA 100 and TA 1537) and at and above 1000 μg/plate (TA 1535, TA 98 and E.coli). In the presence of S9 mix a relevant reduction of revertants was observed at and above 1000 μg/plate (TA 1537 and TA 98) and at and above 2600 μg/plate (TA 1535, TA 100, E.coli). In the PIT bacteriotoxicity (decrease in the number of his+ or trp+ revertants) was observed in the presence of S9 mix in all tester strains at 1000 μg/plate and in tester strain E.coli additionally at 2600 μg/plate. Without S9 mix dependend on the strain at and above 33 μg/plate (TA 1537), at and above 100 μg/plate (TA 100) and at 333 μg/plate (TA 1535, TA 98 and E.coli).
- Individual plate counts, mean number of revertant colonies per plate and standard deviation: yes
- Other: Reduced his- background growth occurred in the tester strain TA 1537 at 5200 μg/Plate in the 1st-SPT and at 333 μg/plate without S9 mix in the 3rd-PIT.

HISTORICAL CONTROL DATA: please refer to Table 1 and 2 in the section "Any other information on materials and methods incl. tables"

Conclusions:

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.

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: 1 μg - 5200 μg/plate (SPT) and 1 μg - 2600 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: Test substance precipitation occurred at 1000 μg/plate and above in all experimental parts with S9 mix and in the 1st SPT without S9 mix.

 

- TOXICITY: Bacteriotoxic effects were observed depending on the strain and test conditions in all experimental parts.

 

- 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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan - Sep 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by Landesamt für Umwelt Rheinland-Pfalz

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:

SOURCE OF TEST MATERIAL
- Source: Not specified
- Lot/batch number of test material: 0020798322
- Expiration date of the lot/batch: 17 Oct 2020
- Purity: 96.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under storage conditions: The stability of the test substance under storage conditions was guaranteed until 17 Oct 2020 as indicated by the sponsor.
- Stability under test conditions: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Due to the use of culture medium (HAM´s F12) as vehicle the verification of the stability of the test substance in the vehicle was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentrations of all suspended test substance concentrations. Homogeneous test substance preparations in the vehicle were prepared individually by shaking and treating with ultrasound. All further concentrations were prepared by serial dilution from the highest dissolved concentration according to the planned doses. All test substance formulations were prepared immediately before administration.

FORM AS APPLIED IN THE TEST: Homogeneous solution

OTHER SPECIFICS
- physical state, appearance: Solid, white
- molecular weight: 435 g/mol

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type of cells: CHO (Chinese hamster ovary) cell line
- Source: cell stock of testing facility
- Suitability of cells: yes, as recommended in OECD TG 476
- Normal cell cycle time (negative control): not specified

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: 3
- Methods for maintenance in cell culture: Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mgfree). Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks. This reaction was stopped by adding 6 mL culture medium incl. 10% (v/v) FCS. Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension. Cells were counted in a counting chamber or using a cell counter. Cell suspensions were diluted with complete culture medium to the desired cell count.
- Doubling time: about 12 - 16 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (stock solution: 250 μg/mL); 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity

Metabolic activation:

with and without

Metabolic activation system:

- type and composition of metabolic activation system: exogenous metabolic activation by cofactor-supplemented postmitochondrial fraction (S9 mix)
- source of S9: liver S9 fraction from phenobarbital- and β-naphthoflavone induced rats
- method of preparation of S9 mix: the S9 mix was prepared freshly prior to each experiment. 1 part S9 fraction was mixed with 9 parts S9 supplement (consisting of 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 15 mM phosphate buffer (pH 7.4))
- concentration or volume of S9 mix and S9 in the final culture medium: 8 mL S9 mix and 32 mL test substance preparation (final volume 40 mL)

Test concentrations with justification for top dose:

- 0; 4.0; 7.1; 12.9; 23.1; 41.7; 75.0; 100.0; 120.0; 200.0 μg/mL (with and without S9 mix)
- The highest tested concentration (200.0 μg/mL) was based on test substance precipitation in culture medium. Dose selection for genotoxicity testing was based on the solubility properties of the test substance in culture medium. Only the test groups 0 - 75 µg/mL were evaluated for gene mutations.

Vehicle / solvent:

- Vehicle used: aqueous solvent (HAM´s F12 culture medium)
- Justification for choice of solvent/vehicle: due to the insufficient solubility of the test substance in the most commonly used vehicles (e.g. DMSO, ethanol, acetone etc.), culture medium was the most suitable. Therefore, culture medium was selected as vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Not necessary since culture medium was used as vehicle

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation (1.25 μg DMBA/mL) -

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Not necessary since culture medium was used as vehicle

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation (400 μg EMS/mL)

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:
- Cell density at seeding: 20x10^6 cells in 40 mL medium/flask
- Test substance added in medium: 0; 4.0; 7.1; 12.9; 23.1; 41.7; 75.0; 100.0; 120.0; 200.0 μg/mL

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20-24 hours
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-9 days
- Selection time: 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7-9 days (cloning efficiency 1); 16 days (cloning efficiency 2)
- Method used: colonies were fixed with methanol and stained with Giemsa
- Selective agent: 6-thioguanine; 10 μg/mL; 6-7 days exposure
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2x10^6 cells from every treatment group were seeded in 20 mL selection medium (175 cm^2 flasks) and the remaining colonies were counted at the end of the selection period
- Criteria for small (slow growing) and large (fast growing) colonies: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Mutant frequency

- OTHER: pH, osmolality, solubility, cell morphology

Rationale for test conditions:

The doses/concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test (experimental conduct with records and documentation in general accordance with the GLP principles).

Evaluation criteria:

- Acceptance criteria: The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative controls should not be less than 50% (with
and without S9 mix).
• The background mutant frequency in the negative controls should be within our historical
negative control data range (95% control limit). Weak outliers can be judged acceptable if
there is no evidence that the test system is not “under control”.
• Concurrent positive controls both with and without S9 mix should induce responses that are
compatible with those generated in the historical positive control data base and produce a
statistically significant increase in mutant frequencies compared with the concurrent
negative control

- Assessment criteria: A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative control
value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated
statistically significant increases without a dose-response relationship may indicate a biological
effect but are not regarded as sufficient evidence of mutagenicity.
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 corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent
vehicle control value and within the range of our laboratory’s historical negative control data
(95% control limit).

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose. The calculation was performed using EXCEL function RGP. The used model is one of the proposed models of the International Workshop on genotoxicity Test procedures Workgroup Report.

A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using EXCEL function HYPGEOM.VERT.

If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

valid

True negative controls validity:

not examined

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 values were not relevantly influenced by test substance treatment.
- Possibility of evaporation from medium: not specified
- Water solubility: not specified
- Precipitation and time of the determination: In all experiments without S9 mix, test substance precipitation was observed macroscopically and microscopically in culture medium at the end of treatment at 75.0 μg/mL and above. In the presence of S9 mix test substance precipitation at the end of treatment occurred at 75 μg/mL and above in the first experiment.
- Definition of acceptable cells for analysis: yes, please refer to section "Evaluation criteria"
- Other confounding effects: In the presence of S9 mix, after 4 hours treatment in the 7th Experiment the morphology and attachment of the cells was adversely influenced (grade > 2) in at least the highest applied concentration (200 μg/mL).

RANGE-FINDING/SCREENING STUDIES: yes

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: There was no relevant decrease in the number of colonies as described by the relative survival in the presence and absence of S9 mix up to the highest evaluated concentrations for gene mutation
o For Relative survival (RS) and cloning efficiency (CE) see section "Any other information on results incl. tables"

- Genotoxicity results:In the 7th Experiment in the absence of metabolic activation, the values for the corrected mutation frequencies ranged between 0.91 – 6.20 per 10^6 cells. The value in the test group 23.1 μg/mL (MFcorr.: 6.20 per 10^6 cells) was statistically significantly increased compared to the concurrent negative control value (MFcorr.: 1.46 per 10^6 cells) and slightly above the range of the 95% control limit. A concentration related increase in the mutant frequencies was, however, not observed. The relevance of the observed statistical significances in the 7th Experiment was assessed in an 8th Experiment. In the 8th Experiment the mutation frequencies of test substance treated cultures ranged between 1.16 – 4.83 per 10^6 cells. All values were within the 95% control limit of the historical control data and not significantly higher
than the concurrent vehicle control group (1.73 mutants per 10^6 cells). A concentration related increase in the mutant frequencies was not observed. To clarify the results of the 7th and 8th Experiment a 9th experiment was carried out. In this experiment the corrected mutation frequencies ranged between 0.00 – 5.88 per 10^6 cells. The value of the corrected mutant frequency of the concurrent negative control was 2.46 per 10^6 cells. Again, all values were within the range of the 95% control limit. A concentration related increase in the mutant frequencies was not observed and no statistically significant increase in the mutant frequencies was determined. In the 7th Experiment in the presence of metabolic activation, the values for the corrected mutation frequencies ranged between 0.68 – 4.00 per 10^6 cells; the respective vehicle control value had 5.06 per 10^6 cells. The obtained values were within the range of the 95% vehicle control limit. A concentration related increase in the mutant frequencies was not observed and no statistically significant increase in the mutant frequencies was determined.
o Number of cells treated and sub-cultures for each cultures: 20x10^6 logarithmically growing cells per flask
o Number of cells plated in selective and non-selective medium: 2x10^6 cells from every treatment group in 20 mL
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: see section "Any other information on results incl. tables"

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see table 1 in section "Any other information on materials and methods incl. tables"
- Negative (solvent/vehicle) historical control data: see table 2 in section "Any other information on materials and methods incl. tables"

Table 1: Cytotoxicity data – 7th Experiment without S9 mix; 4-hour exposure period

 

Test groups [µg/mL]	Cell density at the end of treatment [x 10^5 / mL]	Cloning efficiency 1 (survival) (4 h after treatment; 200 cells/dish seeded)					Relative survival [%]
		Number of colonies		Absolute [%]	Relative [%]	Adjusted [%]
		Dish 1	Dish 2
Negative control	4.73	200	190	97.5	100.0	92.2	100.0
4.0	5.86	162	179	85.3	87.4	99.9	108.3
7.1	5.26	202	175	94.3	96.7	99.2	107.5
12.9	5.56	155	162	79.3	81.3	88.1	95.5
23.1	6.90	156	178	83.5	85.6	115.2	124.9
41.7	5.57	200	199	99.8	102.3	111.1	120.5
75.0	8.17	174	161	83.8	85.9	136.8	148.4
100.0	-	n.c.^1
120.0	-
200.0	-
EMS 400.0	8.75	106	110	54.0	55.4	94.5	102.5

 

 

Table 2: Cytotoxicity data – 7th Experiment with S9 mix; 4-hour exposure period

 

Test groups [µg/mL]	Cell density at the end of treatment [x 10^5 / mL]	Cloning efficiency 1 (survival) (4 h after treatment; 200 cells/dish seeded)					Relative survival [%]
		Number of colonies		Absolute [%]	Relative [%]	Adjusted [%]
		Dish 1	Dish 2
Negative control	6.21	190	200	97.5	100.0	121.1	100.0
4.0	8.11	191	173	91.0	93.3	147.6	121.9
7.1	8.47	144	191	83.8	85.9	141.9	117.2
12.9	8.34	160	193	88.3	90.5	147.2	121.6
23.1	8.39	162	159	80.3	82.3	134.7	111.2
41.7	8.24	135	130	66.3	67.9	109.2	90.2
75.0	8.60	96	72	42.0	43.1	72.2	59.7
100.0	-	n.c.^1
120.0	-
200.0	-
DMBA 1.25	6.77	123	108	57.8	59.2	78.2	64.6

 

 

Table 3: Cytotoxicity data – 8th Experiment without S9 mix; 4-hour exposure period

 

Test groups [µg/mL]	Cell density at the end of treatment [x 10^5 / mL]	Cloning efficiency 1 (survival) (4 h after treatment; 200 cells/dish seeded)					Relative survival [%]
		Number of colonies		Absolute [%]	Relative [%]	Adjusted [%]
		Dish 1	Dish 2
Negative control	5.25	164	189	88.3	100.0	92.7	100.0
4.0	6.79	151	189	85.0	96.3	115.4	124.6
7.1	6.40	193	199	98.0	111.0	125.4	135.4
12.9	6.54	179	191	92.5	104.8	121.0	130.6
23.1	6.53	216	196	103.0	116.7	134.5	145.2
41.7	6.81	218	183	100.3	113.6	136.5	147.4
75.0	6.97	142	181	80.8	91.5	112.6	121.5
100.0	-	n.c.^1
120.0	-
200.0	-
EMS 400.0	7.17	111	124	58.8	66.6	84.2	90.9

 

 

Table 4: Cytotoxicity data – 9th Experiment without S9 mix; 4-hour exposure period

 

Test groups [µg/mL]	Cell density at the end of treatment [x 10^5 / mL]	Cloning efficiency 1 (survival) (4 h after treatment; 200 cells/dish seeded)					Relative survival [%]
		Number of colonies		Absolute [%]	Relative [%]	Adjusted [%]
		Dish 1	Dish 2
Negative control	8.23	157	177	83.5	100.0	137.4	100.0
4.0	9.49	167	195	90.5	108.4	171.8	125.0
7.1	9.26	165	163	82	98.2	151.9	110.5
12.9	9.70	188	165	88.25	105.7	171.2	124.6
23.1	8.79	155	161	79	94.6	138.9	101.0
41.7	7.23	148	160	77	92.2	111.3	81.0
75.0	8.07	137	156	73	87.7	118.2	86.0
100.0	n.c.^1
120.0
200.0
EMS 400.0	6.80	115	109	56	67.1	76.2	55.4

 

 

Table 5: Mutant frequency and viability – 7th Experiment without S9 mix; 4-hour exposure period

 

Test groups [µg/mL]	Cloning efficiency 2 (viability)				Mutant frequency
	Number of colonies		CE2		Number of colonies^a		MF (per 10^6 cells)
	Dish 1	Dish 2	Abs. [%]	Rel. [%]	Flask 1	Flask 2	Uncorrected	Corrected^b
Negative control	188	154	85.5	100.0	5	0	1.25	1.46
4.0	158	158	79.0	92.4	7	8	3.75	4.75
7.1	161	168	82.3	96.2	2	1	0.75	0.91
12.9	207	178	96.3	112.6	4	3	1.75	1.82
23.1	199	188	96.8	113.2	13	11	6.00	6.20^s
41.7	172	151	80.8	94.4	0	7	1.75	2.17
75.0	165	170	83.8	98.0	9	5	3.50	4.18
100.0	n.c.^1
120.0
200.0
EMS 400.0	170	171	85.3	99.7	178	188	91.50	107.33^s

 

 

Table 6: Mutant frequency and viability – 7th Experiment with S9 mix; 4-hour exposure period

 

Test groups [µg/mL]	Cloning efficiency 2 (viability)				Mutant frequency
	Number of colonies		CE2		Number of colonies^a		MF (per 10^6 cells)
	Dish 1	Dish 2	Abs. [%]	Rel. [%]	Flask 1	Flask 2	Uncorrected	Corrected^b
Negative control	174	142	79.0	100.0	5	11	4.00	5.06
4.0	165	177	85.5	108.2	1	2	0.75	0.88
7.1	142	152	73.5	93.0	2	0	0.50	0.68
12.9	164	157	80.3	101.6	2	1	0.75	0.93
23.1	157	168	81.3	102.8	11	2	3.25	4.00
41.7	137	119	64.0	81.0	4	6	2.50	3.91
75.0	155	147	75.5	95.6	2	4	1.50	1.99
100.0	n.c.^1
120.0
200.0
DMBA 1.25	100	88	47.0	59.5	84	92	44.00	93.62^s

 

 

Table 7: Mutant frequency and viability – 8th Experiment without S9 mix; 4-hour exposure period

 

Test groups [µg/mL]	Cloning efficiency 2 (viability)				Mutant frequency
	Number of colonies		CE2		Number of colonies^a		MF (per 10^6 cells)
	Dish 1	Dish 2	Abs. [%]	Rel. [%]	Flask 1	Flask 2	Uncorrected	Corrected^b
Negative control	113	176	72.3	100.0	2	3	1.25	1.73
4.0	n.c.
7.1
12.9	176	149	81.3	112.5	8	6	3.50	4.31
23.1	163	182	86.3	119.4	3	1	1.00	1.16
41.7	122	147	67.3	93.1	5	8	3.25	4.83
75.0	175	139	78.5	108.7	6	8	3.50	4.46
100.0	n.c.^1
120.0
200.0
EMS 400.0	114	140	63.5	87.9	198	191	97.25	153.15^s

 

 

Table 8: Mutant frequency and viability – 9th Experiment without S9 mix; 4-hour exposure period

 

Test groups [µg/mL]	Cloning efficiency 2 (viability)				Mutant frequency
	Number of colonies		CE2		Number of colonies^a		MF (per 10^6 cells)
	Dish 1	Dish 2	Abs. [%]	Rel. [%]	Flask 1	Flask 2	Uncorrected	Corrected^b
Negative control	160	165	81.3	100.0	5	3	2.00	2.46
4.0	143	138	70.3	86.5	4	6	2.50	3.56
7.1	151	148	74.8	92.0	0	0	0.00	0.00
12.9	159	127	71.5	88.0	5	3	2.00	2.80
23.1	153	136	72.3	88.9	8	9	4.25	5.88
41.7	134	171	76.3	93.8	6	6	3.00	3.93
75.0	162	160	80.5	99.1	6	3	2.25	2.80
100.0	n.c.^1
120.0
200.0
EMS 400.0	123	103	56.5	69.5	215	213	107.00	189.38

 

a = number of colonies 7 days after seeding about 2x10^6 cells/flask in selection medium

b = correction on the basis of the absolute cloning efficiency 2 at the end of the expression period

n.c.1 = Culture was not continued since only one concentration beyond the solubility limit is required

S = mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)

Conclusions:

In the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Nine independent experiments were carried out, nine experimental parts without and three experimental parts with the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Due to faulty preparation of the test substance formulations in the experiments 1-4, they were considered as invalid. The 5th Experiment was also invalid due to strong cytotoxicity. The 6th Experiment was not scored, since no relevant cytotoxicity or test substance precipitation was observed up to the highest tested concentration (90 μg/mL) and thus, did not meet the criteria of the highest test concentration of OECD 476. The data of all invalid studies (Exp. 1-6) are not reported. The highest tested concentration (200.0 μg/mL) in the 7th Experiment was based on test substance precipitation in culture medium. Culture medium (HAM´s F12) was used as vehicle. Test groups printed in bold type were evaluated for gene mutations:

7th Experiment:

- Without S9 mix
0; 4.0; 7.1; 12.9; 23.1; 41.7; 75.0; 100.0; 120.0; 200.0 μg/mL

- With S9 mix
0; 4.0; 7.1; 12.9; 23.1; 41.7; 75.0; 100.0; 120.0; 200.0 μg/mL

8th Experiment:

- Without S9 mix
0; 4.0; 7.1; 12.9; 23.1; 41.7; 75.0; 100.0; 120.0; 200.0 μg/mL

9th Experiment:

- Without S9 mix
0; 4.0; 7.1; 12.9; 23.1; 41.7; 75.0; 100.0; 120.0; 200.0 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in  6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.
The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically  significant increase in the frequencies of forward mutations. Dose selection for genotoxicity testing was based on the solubility properties of the test substance in culture medium. At least the highest tested concentrations in the main experiments showed clear test substance precipitates in culture medium macroscopically at the end of exposure period. In the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations  evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

In a reverse mutation assay (accordig to OECD guideline 471 and GLP) the mutagenic potential of the test substance was assessed based on the ability to induce point mutations in selected loci of the bacterial strains S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The test substance was administered in a dose range of 1 μg - 5200 μg/plate in the standard plate test and of 1 μg - 2600 μg/plate in the preincubation test, both with and without metabolic activation (liver S9 mix from induced rats). Test substance precipitation occurred at 1000 μg/plate and above in all experimental parts with S9 mix and in the 1st SPT without S9 mix. Bacteriotoxic effects were observed depending on the strain and test conditions in all experimental parts. 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. 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.

 

HPRT assay:

The test material was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro following OECD guideline 476 (GLP-conform).  Nine independent experiments were carried out, nine experimental parts without and three experimental parts with the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Due to faulty preparation of the test substance formulations in the experiments 1-4, they were considered as invalid. The 5th Experiment was also invalid due to strong cytotoxicity. The 6th Experiment was not scored, since no relevant cytotoxicity or test substance precipitation was observed up to the highest tested concentration (90 μg/mL) and thus, did not meet the criteria of the highest test concentration of OECD 476. The data of all invalid studies (Exp. 1-6) are not reported. The highest tested concentration (200.0 μg/mL) in the 7th Experiment was based on test substance precipitation in culture medium. Culture medium (HAM´s F12) was used as vehicle. Test groups 0 - 75.0 µg/mL were evaluated for gene mutations. The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. Dose selection for genotoxicity testing was based on the solubility properties of the test substance in culture medium. At least the highest tested concentrations in the main experiments showed clear test substance precipitates in culture medium macroscopically at the end of exposure period. In the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

 

Micronucleus test:

According to OECD guideline 487 and GLP, the test item suspended in culture medium, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments with and without S9 mix. In each experimental group two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (2056 µg/mL of the test item) was chosen with regard to the molecular weight and the purity of the test item and with respect to the current guideline. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation. In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In Experiment II in the absence of S9 mix after continuous treatment, clear cytotoxicity (cytostasis of 56.1%) was observed at the highest evaluated concentration. In this study in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008:

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in vitro mammalian Cell Micronucleus Test (OECD 487, GLP). 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.