Reaction mass of butanoic acid, 2-ethyl-2,3,3-trimethyl-, ethyl ester and butanoic acid, 2,3-dimethyl-2-(1-methylethyl)-, ethyl ester and pentanoic acid, 2,2,4,4-tetramethyl-, ethyl ester and ethyl 2,2,3,3-tetramethylpentanoate and ethyl 2,2,3,4-tetramethylpentanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay (OECD guideline 471), in an in vitro mammalian cell micronucleus assay (OECD guideline 487) and in an in vitro mammalian cell gene mutation assay (OECD guideline 490), Neova was not mutagenic or induced chromosome aberrations in the absence and the presence of metabolic activation.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998-08

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: V03 NEOVA 9-Dest. Fraktion 3-25
- Expiration date of the batch: Sept 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Target gene:

his, trp

Species / strain / cell type:

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

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:

- Source of S9: Phenobarbital- and ß-naphthoflavone-induced rat liver S9 mix

- Method of preparation of S9 mix: Male Wistar rats 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 prepared washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (containing 50 µL S9) in a total volume of 2.7 mL

- Quality controls of S9
Sterility: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.
Efficacy: The S9 batch was characterized with benzo[a]pyrene.

Test concentrations with justification for top dose:

1st experiment (plate incorporation): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd experiment (pre-incubation): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate;
In agreement with the recommendations of current guidelines, 5 mg/plate or 5 μL/plate were generally selected as maximum test dose.

Vehicle / solvent:

- Vehicle/solvent used: DMSO (test item and all positive controls)

- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO 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:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

- S9 mix, tested with Escherichia coli WP2 uvrA (5 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

- S9 mix, tested with TA 1537 (100 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

- S9 mix, tested with TA 98 (10 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

- S9 mix, tested with TA 1535, TA 100 (5 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

+ S9 mix, tested with TA 1535, TA 100, TA 1537, TA 98 (2.5 µg/plate) and with Escherichia coli WP2 uvrA (60 µg/plate)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicates (both in the 1st and 2nd experiment)

- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Plate incorporation (1st experiment), pre-incubation (2nd experiment)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes
- Exposure duration/duration of treatment: 48 – 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Background growth inhibition and decrease in the number of revertants (factor ≤ 0.6)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The bacterial colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation 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:

Not performed as not mandatory for this test system.

Key result

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

Key result

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

Key result

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

Key result

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

Key result

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
Solubility: Test substance precipitation was observed at 5000 μg/plate with and without S9 mix.
Sterility: There was no bacterial growth in the sterility controls.

TOXICITY
A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions at and above 1000 μg/plate.
In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions at and above 333 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See section "Attached background material".

Ames test:
- Signs of toxicity: See section "Attached background material".
- Individual plate counts: See section "Attached background material".
- Mean number of revertant colonies per plate and standard deviation: See section "Attached background material".

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016-07-29

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

yes

Remarks:

Differences e.g. in dose selection. The recommendations of the most recent OECD Guideline were followed.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998-08

Deviations:

yes

Remarks:

Differences e.g. in dose selection. The recommendations of the most recent OECD Guideline were followed.

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: V03 NEOVA 9-Dest. Fraktion 3-25
- Expiration date of the batch: Sept 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

clone 3.7.2c

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: derived from a permanent cell line of 3-methylcholanthrene induced tumors (thymus) in DBA/2 mice
- Suitability of cells: high proliferation rate, high plating efficiency, stable karyotype
- Normal cell cycle time (negative control): doubling time of about 9 - 10 hours

For cell lines:
- Absence of Mycoplasma contamination: Each batch used for mutagenicity testing was checked for mycoplasma contaminations
- Methods for maintenance in cell culture: The cells were subcultured twice weekly (routine passage in 75 cm2 flasks). All incubations occurred with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity.
- Cell cycle length, doubling time or proliferation index : doubling time of about 9 - 10 hours
- Modal number of chromosomes: 40 ± 1 chromosomes

MEDIA USED
- Type and composition of media
RPMI-0: RPMI 1640 medium including stable glutamine supplemented with 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL) and 1% (v/v) sodium pyruvate (10 mM)
RPMI-5: RPMI-0 supplemented with 5% (v/v) fetal calf serum
RPMI-10: RPMI-0 supplemented with 10% (v/v) fetal calf serum
RPMI-20: RPMI-0 supplemented with 20% (v/v) fetal calf serum
Pretreatment medium A ("THMG" medium): RPMI-10 supplemented with thymidine, hypoxanthine, methotrexate, glycine
Pretreatment medium B ("THG" medium): RPMI-10 supplemented with thymidine, hypoxanthine, glycine
Selection medium ("TFT" medium): RPMI-20 supplemented with trifluorothymidine

- CO2 concentration, humidity level, temperature: 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: phenobarbital- and ß-naphthoflavone-induced rat liver S9 mix
- Method of preparation of S9 mix: Male Wistar rats 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 prepared washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.8 mL S9 mix (containing 80 µL S9) in a total volume of 20 mL

Test concentrations with justification for top dose:

1st Experiment
with S9 mix (4-hour exposure period)
0; 3.8; 7.5; 15.0; 30.0; 40.0; 50.0; 60.0; 100 μg/mL
3rd Experiment
without S9 mix (4-hour exposure period)
0; 3.8; 7.5; 15.0; 30.0; 40.0; 50.0; 60.0; 100 μg/mL
4th Experiment
without S9 mix (24-hour exposure period)
0; 3.8; 7.5; 15.0; 20.0; 30.0; 40.0; 50.0; 60.0 μg/mL

Dose selection: based on the data and the observations from a pretest and taking into account the current guidelines or recommended criteria.

Vehicle / solvent:

- Vehicle/solvent used: DMSO (test item, 7,12-dimethylbenz[a]anthracene), RPMI-0 medium (methyl methanesulfonate, cyclophosphamide)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water and culture medium, dimethyl sulfoxide (DMSO) was selected as vehicle, which had been demonstrated to be suitable in the mouse lymphoma assay and for which historical data are available. The final concentration of the vehicle DMSO in culture medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

cyclophosphamide

Remarks:

with metabolic activation, 1.0 μg/mL (7,12-dimethylbenz[a]anthracene) and 2.5 μg/mL (cyclophosphamide)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation, 15 μg/mL (4-hour exposure period) and 5 μg/mL (24-hour exposure period)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 4 independent experiments were carried out (+/-S9). In the 1st Experiment without S9 mix, the number of cells (2nd passage) in culture A were very low. Therefore, further cultivating was not possible. Much higher cell densities at the parallel tested B cultures were found. In the 2nd Experiment the assessment of 96-well plates for survival showed that the cloning efficiency could not be made for any of the groups. Due to these technical reasons these experimental parts were invalid and the data were not reported.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 4-hour exposure: 1.5 x 10^7 cells per culture; 24-hour exposure: 1 x 10^7 cells per culture
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Pretreatment period: Spontaneous TK deficient mutants (TK-/-) were eliminated from the stock cultures by incubating cells for 1 day in pretreatment medium A, and for the following 3 days in pretreatment medium B
- Exposure duration/duration of treatment: 4 (with and without S9) and 24 hours (without S9)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time: 9 days
- Method used: microwell plates
- Selective agent: Trifluorothymidine (TFT), 4.0 μg/mL, min. 9 days
- Criteria for small (slow growing) and large (fast growing) colonies: The colonies are classified into large colonies (indication of gene mutation) and small colonies (indication of chromosome breakage). Small colonies are defined as less than 1/4 of the diameter of the well. Size is the key factor and morphology (the optical density of the small colonies is considerably higher) should be secondary.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cloning efficiency 1 (survival): Sampling at the end of the exposure period.
- Cloning efficiency 2 (viability): Sampling after the expression period (2 days after end of exposure).
- Relative suspension growth and relative total growth
For calculation of the relative suspension growth (RSG) and the relative total growth (RTG) the cell counts determined within the expression period at 2nd and 3rd passage after exposure in the case of 4-hour exposure and 1st, 2nd and 3rd passage after exposure in the case of 24- hour exposure were used.

METHODS FOR MEASUREMENTS OF GENOTOXICITY
Mutant frequency
The number of empty wells and the number of wells containing colonies were scored and reported. The colonies are classified into large colonies (indication of gene mutation) and small colonies (indication of chromosome breakage).

Uncorrected mutant frequency
The uncorrected mutant frequency per 10^6 cells (MFuncorr.) was calculated for each test group as follows:
MFuncorr. = ((-ln (total number of empty wells) / (total number of seeded wells)) / (number of seeded cells per well)) x 10^6

Corrected mutant frequency
The corrected mutation frequency (MFcorr.) was calculated regarding the values of CE2:
MFcorr. = (MFuncorr. / CE2) x 100

Determination of borderline mutant frequency based on GEF
The GEF (global evaluation factor) evaluation method requires that the MF exceeds a value based on the global distribution of the background MF of the test method. This value is defined as the mean of the negative/vehicle MF distribution plus one standard deviation. Based on a large data base (n = 493 experiments) from six laboratories a GEF of 126 mutant colonies per 10^6 cells (mean MFcorr = 99 x 10^-6 colonies; standard deviation = 27 x 10^-6 colonies) was calculated for the microwell method. To be judged positive the mutation frequency has to exceed a threshold of 126 colonies per 10^6 cells (GEF) above the concurrent negative/vehicle control value. The borderline mutant frequency was calculated for each experiment separately as follows:
Borderline MF = MFvehicle control corr + GEF (126 x 10^-6)

Evaluation criteria:

The test substance is considered mutagenic if all following criteria are met:
- The mutation frequency exceeds a threshold of 126 mutant colonies per 10^6 cells (GEF: Global Evaluation Factor) above the concurrent negative/vehicle control value.
And
- Evidence of reproducibility of any increase in mutant frequencies, means the mutagenic response occurs at least in both parallel cultures of one experiment.
And
- A statistically significant dose-related increase in mutant frequencies using an appropriate statistical trend test.

The test substance is considered non-mutagenic if at least one of the following criteria is met:
- The mutation frequency is below a threshold of 126 mutant colonies per 10^6 cells (GEF) above the concurrent negative/vehicle control value.
Or
- No evidence of reproducibility of an increase in mutant frequencies is obtained.
Or
- No statistically significant dose-related increase in mutant frequencies using an appropriate statistical trend test is observed.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls (95% control limit) and the mutation rates of all negative and vehicle controls of this study were taken into consideration. Results of test groups were rejected if the RTG was less than 10% of the respective vehicle control. Whenever a test substance is considered mutagenic according to the above-mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose-related shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

An appropriate statistical method to test for linear trend (MS EXCEL function RGP; 10) was performed to assess a possible linear dose-relation in mutant frequencies. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. A trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. However, both, biological and statistical significance have been considered together.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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
- Osmolality: Not relevantly influenced by test substance treatment.
- pH values: Not relevantly influenced by test substance treatment.
- Solubility: No precipitation in culture medium was observed up to the highest applied test substance concentration.

STUDY RESULTS
Gene mutation tests in mammalian cells:
- Genotoxicity results: See section "Attached background material".
No increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. The corrected mutant frequencies obtained for both experiments were always close to the respective vehicle control values and below the calculated threshold taking in consideration the Global Evaluation Factor (GEF).
In the 3rd Experiment after 4 hours treatment in the absence of S9 mix the values for the corrected mutation frequencies (MFcorr.: 56.1 – 72.4 per 10^6 cells) were close to the respective vehicle control value (MFcorr.: 61.5 per 10^6 cells) and clearly within the limit of the mutant frequency threshold (188 per 10^6 cells). The value of the vehicle control group was in the range of the of our 95% limit historical vehicle control data (MFcorr.: 22.7 – 90.5 per 10^6 cells).
In the 4th Experiment after 24 hours treatment in the absence of S9 mix the values for the corrected mutation frequencies (MFcorr.: 66.3 – 93.8 per 10^6 cells) were close to the respective vehicle control value (MFcorr.: 80.6 per 10^6 cells) and within the limit of the mutant frequency threshold (207 per 10^6 cells). The value of the vehicle control group was in the range of our historical vehicle control data (MFcorr.: 13.5 – 88.3 per 10^6 cells).
In the presence of S9 mix after 4 hours treatment the values for the corrected mutation frequencies (MFcorr.: 63.1 – 64.9 per 10^6 cells) were close to the respective vehicle control value (MFcorr.: 63.6 per 10^6 cells) and within the limit of the mutant frequency threshold (190 per 10^6 cells). The value of the vehicle control group was in the range of our historical negative control data (MFcorr.: 22.0 – 94.7 per 10^6 cells).
The statistical analyses by testing for linear trend led to clearly negative findings for all experiments with and without S9 mix.
Both the positive control substances known to induce gene mutations led to clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (MFcorr.: 301.6 – 694.3 per 10^6 cells) clearly exceeded the respective calculated thresholds for a mutagenic effect based on the GEF (126 plus the mutant frequency of the respective vehicle control). In addition, the corrected mutant frequencies were
within our historical positive control data range (222.2 – 1496.6 per 10^6 cells). The obtained values fulfilled the criteria for positive controls as mentioned in the current OECD Guideline 490.

- Results from cytotoxicity measurements: See section "Attached background material".
In both experiments, clear cytotoxic effects indicated by reduced cloning efficiencies or reduced relative total growth of below 20% of control were observed in the presence and in the absence of S9 mix.
In the absence of S9 mix the relative total growth (RTG) ranged between 71.7 - 87.6 % observed from 3.8 – 40.0 μg/mL in the 3rd Experiment using pulse treatment. The doses above 40.0 μg/mL (50; 60 and 100 μg/mL) were strongly cytotoxic and the relative total growth could not be determined. In the 4th Experiment using continuous treatment for 24 hours the RTG ranged between 79.3 - 101.9 % observed from 3.8 – 40.0 μg/mL. The doses above 40.0 μg/mL (50 and 60 μg/mL) were strongly cytotoxic and the relative total growth could not be determined.
In the presence of S9 mix the relative total growth (RTG) ranged between 74.2 - 106.3 % observed from 3.8 – 50.0 μg/mL. Higher doses above 50.0 μg/mL (60 and 100 μg/mL) were strongly cytotoxic and the relative total growth could not be determined. Although using dilution steps of 1.2 or 1.25 in the critical cytotoxicity range, relative total growth values in the range of 10 to 20% were not obtained due to steep test substance cytotoxicity.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

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:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 735/2017; B.49: IN VITRO MAMMALIAN CELL MICRONUCLEUS TEST

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Fresh Blood was collected from a single donor for each experiment. Only healthy, non-smoking donors and not receiving medication were used. In this study, in the 1st and 2nd Experiment blood from female donors (32 and 25 years old, respectively) were used.
- 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.

For lymphocytes:
- Sex, age and number of blood donors: 2 females, 32 and 25 years old
- Whether whole blood or separated lymphocytes were used: Whole blood cultures were used
- Whether blood from different donors were pooled or not: Blood from a single donor was used for each experiment.
- Mitogen used for lymphocytes: Phytohemagglutinin (PHA)

MEDIA USED
- Type and composition of media
Stimulation medium: Medium + 1% P/S +1% HEPES supplemented with 0.5% [v/v] Phytohemagglutinin (PHA, stock solution 0.6 mg/mL, final concentration 3 μg/mL) and 0.5% [v/v] sodium heparin (25000 IE)
Cytochalasin B treatment medium: Medium + 1% P/S +1% HEPES supplemented with 30 μL Cytochalasin B (Cyt B, stock solution: 2 mg/mL in DMSO, final concentration: 6 μg/mL)
Treatment medium (4 hours treatment without and with S9 mix): DMEM/F12 +1% P/S +1% HEPES medium containing stable glutamine
Treatment medium (20 hours treatment without S9 mix): DMEM/F12 medium + 1% P/S +1% HEPES containing stable glutamine and supplemented with 10% [v/v] FCS.

- CO2 concentration, humidity level, temperature: 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

yes; 6 µg/mL Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital- and ß-naphthoflavone-induced rat liver S9 mix

- Method of preparation of S9 mix: Male Wistar rats 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 prepared washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

- Concentration or volume of S9 mix and S9 in the final culture medium: 6 mL S9 mix (containing 0.6 mL of S9) in a total volume of 30 mL.

- Quality controls of S9:
The sterility of the S9 fraction was determined by incubating 1 mL S9 fraction on an agar plate at 37°C for 48 hours. The content of protein was measured with an appropriate method (e.g. Bradford). To check the activity of the S9 fraction a bacterial reverse mutation assay according to Ames et al. was performed with the positive control Benzo(a)pyrene.

Test concentrations with justification for top dose:

1st experiment (4 hours exposure)
without S9 mix: 3.27, 5.88, 10.58, 19.05, 34.29, 61.73, 111.11, 200.00 μg/mL
with S9 mix: 3.27, 5.88, 10.58, 19.05, 34.29, 61.73, 111.11, 200.00 μg/mL;

2nd experiment (20 hours exposure):
without S9 mix: 3.27, 5.88, 10.58, 19.05, 34.29, 61.73, 111.11, 200.00 μg/mL
Based on the observations (e.g. solubility properties) of a previously performed pretest for solubility, 200.0 μg/mL of the test item was used as top concentration in this cytogenetic study.

Vehicle / solvent:

- Vehicle/solvent used: DMSO (test item, cytochalasin B), DMEM/F12 without FCS (mitomycin c, cyclophosphamide), HBSS (colchicine)

- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water and culture medium, DMSO was selected as a vehicle. DMSO had been demonstrated to be suitable in the in vitro micronucleus test and historical control data are available.

- Justification for percentage of solvent in the final culture medium: The final concentration of the vehicle DMSO in culture medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation, 2.50 μg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

Remarks:

Without metabolic activation, 0.05 μg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation, 0.31 μg/mL mitomycin c for 4 hours exposure and 0.04 μg/mL mitomycin c for 20 hours exposure

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2 separately treated flasks. From each flask three slides were prepared.
- Number of independent experiments: 2

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

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 hours stimulation with phytohemagglutinin
- Exposure duration/duration of treatment: 4 and 20 hours
- Harvest time after the end of treatment: 16 hours recovery time (just for 4 hour-treatment), then 20 hours harvest time

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:

- Cytokinesis block: 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 including the stain used: Cells were suspended in 1-2 mL fresh fixative and spread on slides. The slides were dipped in deionized water, the cells were pipetted on the slide and fixed by passing through a flame. The 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 spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The diameter of the micronucleus has to be less than 1/3 of the main nucleus, the micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell, only binucleated cells were scored.

- Criteria for scoring chromosome aberrations: See section "Evaluation criteria".

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cytokinesis-block proliferation index

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The slides were scored microscopically for binucleated 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:

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). 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 (16)) 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:

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

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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
- Data on pH: Osmolality and pH values were not relevantly influenced by test substance treatment.
- Data on osmolality: Osmolality and pH values were not relevantly influenced by test substance treatment.
- Solubility:
In a pre-experiment, test substance precipitation occurred macroscopically at and above 55.6 μg/mL in culture medium (+/- S9).
Precipitation and time of the determination:
without S9 mix: at the end of treatment at 200.00 μg/mL (1st and 2nd Experiment).
with S9 mix: at the end of treatment at and above 61.73 μg/mL (1st Experiment).

RANGE-FINDING/SCREENING STUDIES: Preliminary solubility test:
Based on the observations (e.g. solubility properties) of a pretest for solubility 200.0 μg/mL Neova was used as top concentration in this cytogenetic study.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See section "Attached background material".

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: See section "Attached background material".
In the 1st Experiment without S9 mix excessive cytotoxic effects occurred from 111.11 μg/mL onward (CBPI cytostasis: 63.6 – 65.3%). Therefore, these test groups were not scored for micronucleated cells. Test groups treated with 61.73 μg/mL and lower showed slight to no cytotoxic effects (CBPI cytostasis: 1.0 – 23.2%). Despite the tight dilution factor used in this study (1.8) cytostasis levels higher than 23.2% could be be achieved. Thus, 61.73 μg/mL is considered as the highest concentration, which can be used for mutagenicity evaluation. After 20 hours continuous treatment without S9 mix in the 2nd Experiment, the highest tested concentration (200.00 μg/mL) was not evaluable due to strong cytotoxicity. After treatment with 111.11 μg/mL the cell morphology was altered such that only a small fraction of the cells could be scored. Furthermore, the cell numbers were also reduced. Only one of two cultures was thus evaluable (CBPI cytostasis of the evaluable culture A: 28.9%), the 2nd Culture was not scorable due to strong cytotoxic effects. Only the evaluable culture was evaluated for the occurrence of micronuclei. Test groups treated with lower concentrations showed low to no cytotoxic effects (CBPI cytostasis: -8.4 – 26.9%).
In the presence of S9 mix, no relevantly reduced proliferative activity (CBPI cytostasis: 0.4 – 12.6%) was observed in the test groups scored for cytogenetic damage.

- Genotoxicity results: See section "Attached background material".
After 4 hours treatment without metabolic activation in the 1st Experiment the values of micronucleated cells of all test groups ranged between 0.5 – 0.8%. A statistically significant increase compared to the concurrent vehicle control group (0.6% micronucleated cells) could not be observed. All values were below the 95% upper control limit of the historical negative control data range (0.9% micronucleated cells). the trend analysis showed no dose-related increase in the number of micronucleated cells. In the 2nd Experiment, after 20 hours treatment without S9 mix the values of all test groups (0.3 – 0.5% micronucleated cells) were below or as high as the value of the concurrent vehicle control group (0.5% micronucleated cells). All values were below the 95% upper control limit of the historical negative control data range (0.9% micronucleated cells).
In the presence of S9 mix (1st Experiment) no statistically significant increase compared to the corresponding vehicle control group (0.7% micronucleated cells) could be observed. The values of all test groups (0.7 – 1.2% micronucleated cells) were below or as high as the 95% upper control limit of the historical negative control data range (1.2% micronucleated cells). No positive dose response as assessed by a trend analysis could be observed.
The positive control substances MMC (without S9 mix 0.04 and 0.31 μg/mL), Colchicine (without S9 mix: 0.05 μg/mL) and CPA (with S9 mix; 2.50 μg/mL) induced statistically significantly increased micronucleus frequencies in all independently performed experiments. In this study, in the absence and presence of metabolic activation the frequencies of micronucleated cells (4 h without S9 mix: 12.2% micronucleated cells; 20 h without S9 mix: 4.4% and 3.3% micronucleated cells (MMC and Col, respectively); with S9 mix 3.0% micronucleated cells) were compatible to the historical positive control data range.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay

In the chosen key study for mutagenicity in bacteria according to OECD TG 471 and GLP (BASF 2020; 40M0493/19M302), Neova 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 (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA). The standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) was performed with a dose range of 33 μg - 5000 μg/plate. Precipitation of Neova was observed at 5000 μg/plate with and without S9 mix. A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions at and above 1000 μg/plate. In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions at and above 333 μg/plate. 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. Thus, Neova is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation under the experimental conditions chosen.

In vitro mammalian cell gene mutation assay

In the chosen key study for mutagenicity in mammalian cells in vitro according to OECD TG 490 and GLP (BASF SE 2020; 52M0493/19M310), Neova was tested for its ability to induce gene mutations at the thymidine kinase (TK) locus or structural chromosome aberrations at chromosome 11 in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method. Cells were treated with the test substance for 4 and 24 hours in the absence of metabolic activation and for 4 hours in the presence of metabolic activation. Subsequently, cells were cultured for an expression period of about 48 hours and then cultured in selection medium for another approx. 10 days. According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested (test groups evaluated for gene mutations are marked with *):

4-hour exposure period without S9 mix: 0; 3.8; 7.5*; 15.0*; 30.0*; 40.0*; 50.0; 60.0; 100 μg/mL

4-hour exposure period with S9 mix: 0; 3.8; 7.5; 15.0*; 30.0*; 40.0*; 50.0*; 60.0; 100 μg/mL

24-hour exposure period without S9 mix: 0; 3.8; 7.5; 15.0*; 20.0*; 30.0*; 40.0*; 50.0; 60.0 μg/mL

The number of large and small colonies was determined.

Cytotoxicity indicated by reduced relative total growth (RTG) of below 20% of control was observed in all experiments in the presence and absence of metabolic activation. Based on the results of the present study, Neova did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system. Thus, Neova did not induce forward mutations or structural chromosome aberrations. The negative controls gave mutant frequencies within the range expected for the L5178Y TK+/- mouse lymphoma cell line. All positive controls either for the induction of gene mutations or clastogenicity – methyl methanesulfonate (MMS), cyclophosphamide (CPP) and 7,12-dimethylbenz[a]anthracene (DMBA) - led to the expected increase in the frequencies of forward mutations.

Overall, under the experimental conditions chosen here, the conclusion is drawn that Neova is not a mutagenic substance under in vitro conditions in the mouse lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation.

In vitro mammalian cell micronucleus assay

In the chosen key study for clastogenicity/aneugenicity in vitro according to OECD TG 487 and GLP (BASF SE 2020; 31M0493/19M308), Neova was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro. Two independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding solubility test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiment, the following concentrations were tested (Test groups evaluated for the occurrence of micronuclei are marked with *):

1st Experiment; 4 hours exposure, without S9 mix: 0; 3.27; 5.88; 10.58; 19.05*; 34.29*; 61.73*; 111.11; 200.00 μg/mL

1st Experiment; 4 hours exposure, with S9 mix: 0; 3.27; 5.88; 10.58; 19.05*; 34.29*; 61.73*; 111.11; 200.00 μg/mL

2nd Experiment; 20 hours exposure, without S9 mix: 0; 3.27; 5.88; 10.58; 19.05*; 34.29*; 61.73*; 111.11*; 200.00 μg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.

The vehicle controls (DMSO) 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 absence of metabolic activation, cytotoxicity indicated by reduced cell proliferation occurred after 4 hours pulse and 20 hours continuous treatment. In the presence of S9 mix, no relevantly reduced proliferation index (CBPI) was observed, On the basis of the results of the present study, Neova did not cause any biologically relevant or statistically significant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, Neova 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.

Justification for classification or non-classification

The present data on genetic toxicity do not fulfill the criteria laid down in regulation (EU) 1272/2008 and therefore, a non-classification is warranted.