Sodium 1-hydroxyethanesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A battery of three GLP-compliant in vitro genotoxicity studies according to the current OECD TGs is available for the substance:

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 (40M0540/16M386, 2017). 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. The substance was therefore considered to be non-mutagenic in the bacterial reverse mutation assay.

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster (50M0540/16X362, 2017). The substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, it is considered to be non-mutagenic in this HPRT assay.

The test item, dissolved in deionized water, was assessed for its potential to induce micronuclei in human lymphocytesin vitroin the absence and presence of metabolic activation by S9 mix (31M0540/16X361, 2017). In the absence of S9 mix, the substance caused statistically significant increases in the number of micronucleated cells and is considered to be mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration. No such increase was observed in the presence of S9 mix.

To assess the in vivo relevance of the positive finding in the in vitro micronucleus test, an in vivo micronucleus test according to OECD TG 474 is proposed.

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)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Content: 50.7 g/100 g sodium 1-hydroxyethanesulphonate
water content: 46.1 g/100 g
(see analytical report, study code 17L00015)
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
Storage stability: The stability of the test substance under storage conditions throughout the study period is guaranteed until 13 Dec 2017 as indicated by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.

Target gene:

his- (E. coli), trp- (S. typhimurium)

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:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

33 μg - 10000 μg/plate (SPT)
33 μg - 10000 μg/plate (PIT)
Due to the purity of the test substance 10.0 mg/plate was used as top dose in all experiments.

Vehicle / solvent:

Due to the good solubility of the test substance in water, water was used as vehicle.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (with S9, TA 1535, TA 100, TA 1537, TA 98); N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (without S9, TA 1535, TA 100);

Details on test system and experimental conditions:

TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 109 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strains mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015.

Salmonella typhimurium
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined. The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances. The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by
the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

Escherichia coli
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions (5). The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Checking the tester strains
The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid). E. coli WP2 uvrA was checked for UV sensitivity. Histidine and tryptophan auxotrophy was checked in each experiment via the spontaneous rate.

EXOGENOUS METABOLIC ACTIVATION

S9 fraction
The S9 fraction was prepared according to Ames et al. (1, 2) at BASF SE in an AAALAC approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 30 - 70%. The day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and 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.

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 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer (6) is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1. To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.

DOSES
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. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, due to the purity of the test substance 10.0 mg/plate was used as top dose in all experiments.

TEST SUBSTANCE PREPARATIONS
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in water. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before administration.

ANALYSIS OF TEST SUBSTANCE PREPARATION
The stability of the test substance (aqueous solution) throughout the study period is guaranteed as indicated by the sponsor, therefore the stability of the test substance in the vehicle water was expected.

EXPERIMENTAL PROCEDURE

Choice of the vehicle
Due to the good solubility of the test substance in water, water was used as vehicle.

Mutagenicity tests

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al..
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The 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 hinders the counting using the Image Analysis System.

• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The 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 hinders the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al..
0.1 mL test solution or vehicle, 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. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The 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.

Scope of tests and test conditions
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 3333 and 10000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control
2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 3333 and 10000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

Species / strain:

bacteria, other: Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the preincubation assay bacteriotoxicity (slight decrease in the number of trp+ revertants) was observed only using the tester strain E.coli without S9 mix at a concentration of 10000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the experimental conditions chosen here, it is concluded that Rongalit 2 PH-B lq. Trilon-free (PBG 10063730) is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance Rongalit 2 PH-B lq. Trilon-free (PBG 10063730) 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.

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 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 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

other: Gene mutation assay in mammalian cells

Target gene:

HPRT assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

1.3-2748 μg/mL
The highest concentration was chosen with respect to the current OECD guideline 476 (2016) regarding the content of the test item (53.9 g/100 g).

Vehicle / solvent:

deionized water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Test Item Preparation
On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10% (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
All formulations were prepared freshly before treatment and used within two hours of preparation. The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiments without metabolic activation.

Analysis of Test Substance Preparation
Due to the composition of the test substance (water content: 46.1 g/100 g) the stability of the test substance in the vehicle water (= deionized water) throughout the study period was guaranteed.

Test System and Supporting Information
Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
Cell Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×106 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
Culture Medium
For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During 4 hours treatment no FBS was added to he medium. During 24 hours treatment the medium was supplemented with 10% FBS. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

PBS
The PBS was composed as follows (per litre):
NaCl 8000 mg
KCl 200 mg
KH2PO4 200 mg
Na2HPO4 150 mg

Saline G
The "saline G" solution had the following constituents (per litre):
NaCl 8000 mg
KCl 400 mg
Glucose•H2O 1100 mg
Na2HPO4•2H2O 192 mg
KH2PO4 150 mg
The pH was adjusted to 7.2.

Mammalian Microsomal Fraction S9 Mix
Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system is necessary.
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. The S9 was prepared and stored according to the currently valid version of the Envigo SOP for rat liver S9 preparation. 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.
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).
The protein concentration of the S9 preparation was 34.9 mg/mL (Lot. No.: 020217) in the pre-experiment and in the main experiments.

Experimental Design and Study Conduct

Pre-experiment
Two pre-experiments were performed in order to determine the toxicity of the test item (4 hours treatment with and without metabolic activation, second pre-test 24 hours treatment without metabolic activation). The pH-value and the osmolarity was measured in both pre-experiments. The general culturing and experimental conditions in the pre-experiments were the same as described below for the mutagenicity experiment.
In the pre-experiments approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (with and without metabolic activation), and for 24 hours (without metabolic activation) (duplicate cultures per concentration level).

Immediately after treatment the test item is removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item was evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks was about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment. In both pre-tests test item concentrations between 54.7 μg/mL and 2748 μg/mL were used. The highest concentration was chosen with respect to the current OECD guideline 476 (2016) regarding the content of the test item (53.9 g/100 g). In the first pre-test no relevant cytotoxic effects, indicated by a relative cloning efficiency of approx. 50% or below were observed after 4 hours treatment up to the highest concentration with and without metabolic activation. In the second pre-test relevant cytotoxic effects occurred after 24 hours treatment at the lowest concentration and above.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 and 24 hours) prior to removal to the test item. No precipitation or phase separation was noted after 4 and 24 hours treatment with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

Dose Selection
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation or phase separation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The concentration range of experiment I was chosen according to the data generated in the pre-experiment. The concentration range of experiment II was chosen based on the results of the second pre-experiment (without metabolic activation) and of the first main experiment (with metabolic activation). To further investigate the results achived in experiment II the experimental part without metabolic activation (24 hours treatment) was repeated using slightly modified concentrations. In the main experiments the individual concentrations were generally spaced by a factor of approximately 1.75 – 3.0.

Experimental Performance
Seeding
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×107 cells were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
Treatment
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS (experiment I with and without S9 mix and experiment II with S9 mix). In the second experiment without S9 mix, the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10% FBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×106 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Data Recording
The data generated were recorded in the raw data. The results are presented in tabular form, including experimental groups with the test item, solvent, and positive controls.

Data Evaluation
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled (see 4.6.2 Dose Selection).
4.8.2 Evaluation of Results
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

Statistical Analysis
A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated 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.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment II relevant cytotoxic effects (adjusted cloning efficiency I <50%) at >=25.4 μg/mL without S9, in experiment IIA at >=305.0 μg/mL. Recomm. tox. range of 10-20% rel. adj. clon. eff. I was covered in experiment II without metabolic activation.

Vehicle controls validity:

valid

Positive controls validity:

valid

Mutagenicity data (Mutation rates), experiment I, culture I

	conc.	P/	S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 106cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water		-	-	5	5	3	6	1	4.0	2.0	15.2
Positive control with EMS	298.0	-	-	51	61	65	49	59	57.0	6.8	222.1
Test item	95.7	-	-	culture was not continued#
Test item	167.4	-	-	culture was not continued#
Test item	293.0	-	-	4	8	4	1	4	4.2	2.5	14.8
Test item	512.7	-	-	5	5	1	2	8	4.2	2.8	16.7
Test item	897.3	-	-	12	12	6	10	8	9.6	2.6	37.2
Test item	1570.3	-	-	4	3	7	7	8	5.8	2.2	20.2
Test item	2748.0	-	-	3	6	6	4	2	4.2	1.8	13.7
Solvent control with water		-	+	3	4	3	4	13	5.4	4.3	14.8
Positive control with DMBA	2.3	-	+	50	44	44	52	55	49.0	4.9	162.9
Test item	95.7	-	+	culture was not continued#
Test item	167.4	-	+	culture was not continued#
Test item	293.0	-	+	11	6	7	8	8	8.0	1.9	21.5
Test item	512.7	-	+	3	8	4	4	5	4.8	1.9	16.2
Test item	897.3	-	+	7	7	10	4	6	6.8	2.2	20.1
Test Item	1570.3	-	+	6	6	3	1	4	4.0	2.1	15.4
Test item	2748.0	-	+	5	4	4	11	7	6.2	2.9	23.3

# culture was not continued as a minimum of only four analysable concentrations is required

Mutagenicity data (Mutation rates), experiment I, culture II

	conc.	P/	S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 106cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water		-	-	3	4	3	9	5	4.8	2.5	20.4
Positive control with EMS	298.0	-	-	66	61	50	55	69	60.2	7.8	208.3
Test item	95.7	-	-	culture was not continued#
Test item	167.4	-	-	culture was not continued#
Test item	293.0	-	-	9	8	9	8	7	8.2	0.8	38.2
Test item	512.7	-	-	6	5	7	8	3	5.8	1.9	35.0
Test item	897.3	-	-	4	6	5	5	11	6.2	2.8	28.6
Test item	1570.3	-	-	7	5	7	9	9	7.4	1.7	38.0
Test item	2748.0	-	-	1	2	3	8	4	3.6	2.7	14.0
Solvent control with water		-	+	3	4	4	7	5	4.6	1.5	21.0
Positive control with DMBA	2.3	-	+	50	48	50	47	55	50.0	3.1	260.3
Test item	95.7	-	+	culture was not continued#
Test item	167.4	-	+	culture was not continued#
Test item	293.0	-	+	9	1	6	7	6	5.8	2.9	31.7
Test item	512.7	-	+	11	7	4	7	8	7.4	2.5	38.1
Test item	897.3	-	+	3	4	3	8	6	4.8	2.2	29.2
Test Item	1570.3	-	+	2	1	7	5	1	3.2	2.7	21.9
Test item	2748.0	-	+	2	5	4	2	6	3.8	1.8	24.0

# culture was not continued as a minimum of only four analysable concentrations is required

Mutagenicity data (Mutation rates), experiment II, culture I

	conc.	P/	S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 106cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water		-	-	7	8	9	6	6	7.2	1.3	24.0
Positive control with EMS	300.0	-	-	114	97	121	93	135	112.0	17.3	437.5
Test item	3.18	-	-	culture was not continued#
Test item	6.36	-	-	culture was not continued#
Test item	12.7	-	-	culture was not continued#
Test item	25.4	-	-	6	4	7	6	4	5.4	1.3	17.4
Test item	50.9	-	-	4	9	10	4	10	7.4	3.1	25.2
Test item	101.8	-	-	8	1	6	8	7	6.0	2.9	18.5
Test item	305.3	-	-	9	8	6	6	11	8.0	2.1	23.1
Test item	916.0	-	-	10	3	7	6	7	6.6	2.5	29.0
Test item	2748.0	-	-	5	8	8	11	8	8.0	2.1	22.2
Solvent control with water		-	+	4	3	7	3	1	3.6	2.2	14.4
Positive control with DMBA	2.3	-	+	31	26	24	27	24	26.4	2.9	108.6
Test item	167.4	-	+	culture was not continued#
Test item	293.0	-	+	5	8	7	7	9	7.2	1.5	29.5
Test item	512.7	-	+	3	5	3	6	4	4.2	1.3	19.8
Test item	897.3	-	+	2	4	7	6	5	4.8	1.9	20.3
Test item	1570.3	-	+	5	10	8	8	8	7.8	1.8	28.5
Test Item	2748.0	-	+	3	4	6	3	4	4.0	1.2	19.7

# culture was not continued as a minimum of only four analysable concentrations is required

Mutagenicity data (Mutation rates), experiment II, culture II

	conc.	P/	S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 106cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water		-	-	10	9	11	8	7	9.0	1.6	21.9
Positive control with EMS	300.0	-	-	120	137	126	115	131	125.8	8.7	325.2
Test item	3.18	-	-	culture was not continued#
Test item	6.36	-	-	culture was not continued#
Test item	12.7	-	-	culture was not continued#
Test item	25.4	-	-	8	5	6	12	6	7.4	2.8	16.4
Test item	50.9	-	-	7	4	7	5	4	5.4	1.5	11.6
Test item	101.8	-	-	6	8	12	6	9	8.2	2.5	20.4
Test item	305.3	-	-	12	9	12	14	4	10.2	3.9	26.8
Test item	916.0	-	-	6	8	8	4	8	6.8	1.8	19.0
Test item	2748.0	-	-	6	5	4	10	11	7.2	3.1	19.1
Solvent control with water		-	+	4	2	2	3	6	3.4	1.7	15.0
Positive control with DMBA	2.3	-	+	19	24	21	31	21	23.2	4.7	88.7
Test item	167.4	-	+	culture was not continued#
Test item	293.0	-	+	5	8	7	10	7	7.4	1.8	35.5
Test item	512.7	-	+	3	9	3	2	3	4.0	2.8	16.2
Test item	897.3	-	+	4	7	3	3	5	4.4	1.7	18.8
Test item	1570.3	-	+	6	6	12	5	4	6.6	3.1	23.3
Test Item	2748.0	-	+	culture was not continued##

# culture was not continued as a minimum of only four analysable concentrations is required

## culture accidentally not continued

Mutagenicity data (Mutation rates), experiment IIA, culture I

	conc.	P/	S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 106cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water		-	-	4	5	5	3	3	4.0	1.0	15.7
Positive control with EMS	300.0	-	-	39	42	51	36	29	39.4	8.1	202.6
Test item	1.3	-	-	1	2	2	3	2	2.0	0.7	9.3
Test item	3.8	-	-	6	5	6	7	7	6.2	0.8	26.4
Test item	11.3	-	-	6	6	2	7	7	5.6	2.1	28.2
Test item	33.9	-	-	culture was not continued#
Test item	102.0	-	-	culture was not continued#
Test item	305.0	-	-	3	2	1	2	3	2.2	0.8	9.5
Test item	916.0	-	-	5	6	6	3	4	4.8	1.3	22.9
Test item	2748.0	-	-	8	5	6	4	5	5.6	1.5	32.1

# culture was not continued as a minimum of only four analysable concentrations is required

Mutagenicity data (Mutation rates), experiment IIA, culture II

	conc.	P/	S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 106cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water		-	-	3	3	4	1	5	3.2	1.5	13.7
Positive control with EMS	300.0	-	-	36	38	42	50	29	39.0	7.7	120.4
Test item	1.3	-	-	4	2	1	2	3	2.4	1.1	8.3
Test item	3.8	-	-	4	2	4	2	3	3.0	1.0	6.7
Test item	11.3	-	-	6	3	5	7	8	5.8	1.9	27.9
Test item	33.9	-	-	culture was not continued#
Test item	102.0	-	-	culture was not continued#
Test item	305.0	-	-	5	3	3	2	1	2.8	1.5	9.4
Test item	916.0	-	-	3	5	3	3	4	3.6	0.9	14.0
Test item	2748.0	-	-	4	7	6	8	8	6.6	1.7	35.7

# culture was not continued as a minimum of only four analysable concentrations is required

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Rongalit 2 PH-B lq. Trilon-free (PBG 10063730) is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item Rongalit 2 PH-B lq. Trilon-free (PBG 10063730) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The assay was performed in three independent experiments, using two parallel cultures each.

The first main experiment was performed with a treatment period of 4 hours with and without microsomal activation. The second main experiment was performed with a 24 hours treatment period without microsomal activation and a 4 hours treatment period with microsomal activation.

In Experiment II without metabolic activation all evaluated concentrations showed cytotoxic effects indicated by a relative adjusted cloning efficiency below 40%. To cover the required cytotoxic range (evaluation of cytotoxic and non cytotoxic concentrations) a repeat experiment (Experiment IIA) was performed with adjusted concentrations.

No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred in experiment I up to the highest concentration with and without metabolic activation. In experiment II relevant cytotoxic effects as described above were noted at 25.4 μg/mL and above without metabolic activation. In experiment IIA relevant cytotoxic effect occurred at 305.0 μg/mL and above. The recommended toxic range of approximately 10-20% relative adjusted cloning efficiency I was covered in experiment II without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiment up to the maximum concentration.

In experiment I without metabolic activation the 95% confidence interval was exceeded at 897.3 μg/mL in culture I, and at 293.0, 512.7, and 1570.3 μg/mL in culture II. In experiment I with metabolic activation the confidence interval was exceeded in culture II at 293.0 and 512.7 μg/mL. In experiment II the confidence interval was exceeded at 305.3 μg/mL in culture II and at 916.0 μg/mL in culture I without metabolic activation, and in both cultures at 293.0 μg/mL with metabolic activation. In experiment IIA the confidence interval was exceeded in both cultures at 11.3 μg/mL and 2748.0 μg/mL without metabolic activation.

A t-test evaluating the data of both parallel cultures showed a significant response in experiment I at 897.3 and 1570.3 μg/mL without metabolic activation, in experiment II at 293.0 μg/mL with metabolic activation, and in experiment IIA at 11.3 μg/mL and 2748.0 μg/mL without metabolic activation.

The linear regression analysis was negative in experiment I, II, and IIA with and without metabolic activation indicating no concentration related effect. Taken together the statistical analysis revealed no biological relevant effect, neither in experiment I nor in experiment II and IIA.

In the main experiments with and without S9 mix the range of the solvent controls was from 13.7 up to 24.0 mutants per 106 cells; the range of the groups treated with the test item was from 6.7 up to 38.2 mutants per 106 cells.

EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 April - 14 September 2017

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

BASF Test substance number: 16/0540-1
Batch: Lab-sample from Dec 2016
CAS number: 918-04-7
Content*: 53.9 g/100 g (100 g/100 g minus water content)
Identity: confirmed
Homogeneity: given
Expiry Date: 13 December 2017
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Additional Test Substance Information
Storage Conditions: At room temperature
Physical state / Appearance: Liquid, colorless to yellowish
Molecular weight: 148.1 g/mol
Stability in Solvent: Stable in drinking/purified water over 24 hours at room temperature.
*Correction for content (minus water content) was made

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication. Blood was collected from a male donor (23 years old) for Experiment I and IIA and from a different male donor (23 years old) for Experiment IIB.
- Suitability of cells: 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.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) (with 200 mM GlutaMAX™), supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS, 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL); CO2 concentration during incubations: 5,5 %

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (supplemented with cofactors) derived from phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

17.9, 31.2, 54.7, 95.7, 167, 293, 513, 897, 1570, 2748 µg/mL
With regard to the molecular weight and the content (53.9%) of the test item, 2748 µg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 17.9 to 2748 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Using a Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2748 µg/mL were chosen as top treatment concentration for Experiment IIA (invalid due to invalid solvent controls) and IIB.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water (10 % final concentration in medium)
- Justification for choice of solvent/vehicle: solubility properties relative non-toxicity to the cell cultures

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 10.0% deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine (continous treatment without met. act.; 150 ng/mL (Exp. IIA); 100 ng/mL (Exp. IIB))

Details on test system and experimental conditions:

Reason for the choice of human lymphocytes:
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.

Cell cultures:
- Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (23 years old) for Experiment I and IIA and from a different male donor (23 years old) for Experiment IIB. 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.

Culture conditions:
- Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was 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).

Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately
2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohaemagglutinin (PHA) to the culture medium for a period of 48 hours.
- Exposure duration: 4 h (pulse exposure; with and without S9 mix); 20 h (continuous exposure, without S9 mix)
- Expression time/recovery period (cells in growth medium): 16 h (only for pulse exposure)
- Selection time (incubation with cytochalasin B (4 µg/mL)): approx. 20 h
- The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time) (40 h after beginning of treatment). Any specific cell cycle time delay induced by the test item was not accounted for directly.
- All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL "saline G" and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: 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.

DETERMINATION OF CYTOTOXICITY
- Method: To describe a cytotoxic effect the Cytokinesis-block proliferation index (CBPI) was determined in 500 cells per culture in dublicate and cytotoxicity was expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
− the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.

A test item can be classified as clastogenic and aneugenic if:
− the number of micronucleated cells is not in the range of the historical laboratory control data and
− either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.

An increase in the number of micronucleated mononucleate cells may indicate that the test item has aneugenic potential.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 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.

Species / strain:

lymphocytes: human primary

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not observed
- Effects of osmolality: not observed
- Precipitation: No precipitation of the test item in the culture medium was observed.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 0.15 – 1.65 % (pulse treatment; without S9 mix); 0.05 – 1.35 % (continuous treatment; without S9 mix); 0.15 – 1.30 % (pulse treatment; with S9 mix)
- Negative (solvent/vehicle) historical control data: 4.15 – 30.30 % (pulse treatment; without S9 mix); 2.10 – 7.25 % (continuous treatment; without S9 mix); 2.10 – 11.90 % (pulse treatment; with S9 mix)
for details see tables 21 - 24

The test item, dissolved in deionized water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Three independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment IIA and IIB, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. At least 1000 binucleate cells per culture were evaluated for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 2748 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the content (53.9%) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

In this study, no precipitation of the test item in the culture medium was observed.

No relevant influence on osmolarity or pH was observed.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied and evaluated concentration.

In Experiment IIA in the absence of S9 mix, one statistically significant increase in the number of micronucleated cells (1.90 %) was observed after treatment with 897 µg/mL. All values (1.90, 1.55, 1.15 %) clearly exceeded the range of the historical control data (0.12 – 1.03 %), but no dose-dependency was observed. In Experiment IIB in the absence of S9 mix, all evaluated concentrations (897, 1570 and 2748 µg/mL) showed a statistically significant increase in the number of micronucleated cells (1.95, 1.55 and 2.35 %) in comparison to the solvent control. All three values clearly exceeded the range of the historical control data (0.12 – 1.03 %).

In Experiment I in the presence of S9 mix, one statistically significant increase in the number of micronucleated cells (0.95 %) was observed after treatment with 1570 µg/mL. Since the value is clearly within the range of the historical control data (0.16 – 1.08 %) and no dose-dependency was observed, the finding can be regarded as biologically irrelevant. In Experiment IIA in the presence of S9 mix, statistically significant increases in the number of micronucleated cells were observed after treatment with 1570 and 2748 µg/mL (1.00 and 1.18 %) in a dose dependend manner. The value of the highest applied concentration slightly exceeded the range of the historical control data (0.16 – 1.08 %). In Experiment IIB in the presence of S9 mix, the solvent control (1.10 % micronucleated cells) slightly exceeded the range of the historical control data (0.16 – 1.08 %), but is clearly within the min-max range of the historical control data (0.15 – 1.30 %). The values of two highest evaluated concentrations (1.20 and 1.65 % micronucleated cells) exceeded the range of the historical control data (0.16 – 1.08 %). The highest evaluated concentration exceeded the min-max range of the historical control data (0.15 – 1.30 %), as well.

Demecolcine (100 and 150 ng/mL), MMC (1.2 µg/mL) or CPA (15.0 and 17.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

TABLES

Table3     Toxicity – Experiment I

Concentration (µg/mL)	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix
Solvent control	4 hrs	40 hrs	1.99	-
17.9	4 hrs	40 hrs	n.p.	n.p.
31.2	4 hrs	40 hrs	n.p.	n.p.
54.7	4 hrs	40 hrs	n.p.	n.p.
95.7	4 hrs	40 hrs	n.p.	n.p.
167	4 hrs	40 hrs	2.06	n.c.
293	4 hrs	40 hrs	2.08	n.c.
513	4 hrs	40 hrs	2.09	n.c.
897	4 hrs	40 hrs	2.11	n.c.
1570	4 hrs	40 hrs	2.10	n.c.
2748	4 hrs	40 hrs	2.12	n.c.
With S9 mix
Solvent control	4 hrs	40 hrs	2.00	-
17.9	4 hrs	40 hrs	n.p.	n.p.
31.2	4 hrs	40 hrs	n.p.	n.p.
54.7	4 hrs	40 hrs	n.p.	n.p.
95.7	4 hrs	40 hrs	n.p.	n.p.
167	4 hrs	40 hrs	2.01	n.c.
293	4 hrs	40 hrs	2.06	n.c.
513	4 hrs	40 hrs	2.05	n.c.
897	4 hrs	40 hrs	2.07	n.c.
1570	4 hrs	40 hrs	2.00	n.c.
2748	4 hrs	40 hrs	2.01	n.c.

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*      Mean value of two cultures
n.p.  Not prepared
n.c.  Not calculated as the CBPI was equal or higher than solvent control value

Table4     Toxicity – Experiment IIA

Concentration (µg/mL)	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix
Solvent control	20 hrs	40 hrs	2.13	-
293	20 hrs	40 hrs	2.15	n.c.
513	20 hrs	40 hrs	2.16	n.c.
897	20 hrs	40 hrs	2.12	0.9
1570	20 hrs	40 hrs	2.07	5.1
2748	20 hrs	40 hrs	2.01	10.9
With S9 mix
Solvent control	4 hrs	40 hrs	2.24	-
293	4 hrs	40 hrs	2.20	3.1
513	4 hrs	40 hrs	2.22	1.2
897	4 hrs	40 hrs	2.26	n.c.
1570	4 hrs	40 hrs	2.22	1.0
2748	4 hrs	40 hrs	2.27	n.c.

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*      Mean value of two cultures
n.c.  Not calculated as the CBPI was equal or higher than solvent control value

Table5     Toxicity – Experiment IIB

Concentration (µg/mL)	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix
Solvent control	20 hrs	40 hrs	1.91	-
293	20 hrs	40 hrs	1.96	n.c.
513	20 hrs	40 hrs	1.84	7.5
897	20 hrs	40 hrs	1.87	4.4
1570	20 hrs	40 hrs	1.75	17.3
2748	20 hrs	40 hrs	1.67	25.6
With S9 mix
Solvent control	4 hrs	40 hrs	1.96	-
293	4 hrs	40 hrs	1.92	4.4
513	4 hrs	40 hrs	1.85	11.7
897	4 hrs	40 hrs	1.98	n.c.
1570	4 hrs	40 hrs	1.86	10.7
2748	4 hrs	40 hrs	1.93	3.0

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*      Mean value of two cultures
n.c.  Not calculated as the CBPI was equal or higher than solvent control value

 

Table6     Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs without S9 mix, Experiment I

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	10 %	-	4 / 40 hrs	102	308	90	1.98	92	315	92	2.00	1.99
Pos. control#^#	1.2 µg	-	4 / 40 hrs	300	184	16	1.43	297	181	22	1.45	1.44	55.4
Test item	897 µg	-	4 / 40 hrs	82	311	107	2.05	53	307	140	2.17	2.11	n.c.
''	1570 µg	-	4 / 40 hrs	84	298	118	2.07	71	290	139	2.14	2.10	n.c.
''	2748 µg	-	4 / 40 hrs	73	307	120	2.09	73	285	142	2.14	2.12	n.c.

*         c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#             Deionised water

^##        MMC

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

 

Table7     Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs with S9 mix, Experiment I

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	10 %	+	4 / 40 hrs	104	301	95	1.98	84	324	92	2.02	2.00
Pos. control#^#	17.5 µg	+	4 / 40 hrs	196	251	53	1.71	159	276	65	1.81	1.76	23.6
Test item	897 µg	+	4 / 40 hrs	72	315	113	2.08	71	330	99	2.06	2.07	n.c.
''	1570 µg	+	4 / 40 hrs	83	323	94	2.02	94	320	86	1.98	2.00	n.c.
''	2748 µg	+	4 / 40 hrs	92	316	92	2.00	78	335	87	2.02	2.01	n.c.

*         c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#             Deionised water

^##        CPA

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

Table8     Number of micronucleated cells; exposure period 4 hrs without S9 mix, Experiment I

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	10 %	-	4 / 40 hrs	0	0	0	0	0	0	0	0	0	0.00
Pos. control#^#	1.2 µg	-	4 / 40 hrs	20	3	1	24	26	2	0	28	52	2.60
Test item	897 µg	-	4 / 40 hrs	2	0	0	2	0	0	0	0	2	0.10
''	1570 µg	-	4 / 40 hrs	1	0	0	1	1	0	0	1	2	0.10
''	2748 µg	-	4 / 40 hrs	0	0	0	0	1	0	0	1	1	0.05

^#             Deionised water

^##           MMC

Table9     Number of micronucleated cells; exposure period 4 hrs with S9 mix, Experiment I

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	10 %	+	4 / 40 hrs	6	0	0	6	2	0	0	2	8	0.40
Pos. control#^#	17.5 µg	+	4 / 40 hrs	56	4	0	60	59	3	0	62	122	6.10
Test item	897 µg	+	4 / 40 hrs	1	1	0	2	5	0	0	5	7	0.35
''	1570 µg	+	4 / 40 hrs	6	0	0	6	12	1	0	13	19	0.95
''	2748 µg	+	4 / 40 hrs	5	0	0	5	6	0	0	6	11	0.55

^#             Deionised water

^##           CPA

 

 

Table10   Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 20 hrs without S9 mix, Experiment IIA

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	10.0 %	-	20 / 40 hrs	44	356	100	2.11	29	367	104	2.15	2.13
Pos. control#^#	150 ng	-	20 / 40 hrs	155	318	27	1.74	170	315	15	1.69	1.72	36.6
Test item	897 µg	-	20 / 40 hrs	31	391	78	2.09	38	350	112	2.15	2.12	0.9
''	1570 µg	-	20 / 40 hrs	52	361	87	2.07	55	352	93	2.08	2.07	5.1
''	2748 µg	-	20 / 40 hrs	57	391	52	1.99	36	415	49	2.03	2.01	10.9

*         c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#             Deionized water

^##        Demecolcine

Table11   Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs with S9 mix, Experiment IIA

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	10.0 %	+	4 / 40 hrs	55	265	180	2.25	53	284	163	2.22	2.24
Pos. control#^#	15.0 µg	+	4 / 40 hrs	195	227	78	1.77	186	220	94	1.82	1.79	36.0
Test item	897 µg	+	4 / 40 hrs	38	285	177	2.28	49	278	173	2.25	2.26	n.c.
''	1570 µg	+	4 / 40 hrs	42	299	159	2.23	52	290	158	2.21	2.22	1.0
''	2748 µg	+	4 / 40 hrs	63	248	189	2.25	49	263	188	2.28	2.27	n.c.

*         c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#             Deionized water

^##        CPA

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

Table12   Number of micronucleated cells; exposure period 20 hrs without S9 mix, Experiment IIA

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	10.0 %	-	20 / 40 hrs	9	0	0	9	9	0	0	9	18	0.90
Pos. control#^#	150 ng	-	20 / 40 hrs	22	2	0	24	19	6	4	29	53	2.65
Test item	897 µg	-	20 / 40 hrs	16	0	1	17	20	1	0	21	38	1.90
''	1570 µg	-	20 / 40 hrs	15	1	0	16	13	2	0	15	31	1.55
''	2748 µg	-	20 / 40 hrs	15	0	0	15	8	0	0	8	23	1.15

^#             Deionized water

^##           Demecolcine

Table13   Number of micronucleated cells; exposure period 4 hrs with S9 mix, Experiment IIA

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	10.0 %	+	4 / 40 hrs	4	0	0	4	4	0	0	4	8	0.40
Pos. control#^#	15.0 µg	+	4 / 40 hrs	61	7	3	71	76	7	1	84	155	7.75
Test item	897 µg	+	4 / 40 hrs	10	0	0	10	5	0	0	5	15	0.75
''	1570 µg	+	4 / 40 hrs	19	1	0	20	20	0	0	20	40*	1.00
''	2748 µg	+	4 / 40 hrs	15	1	0	16	28	3	0	31	47*	1.18

^#             Deionized water

^##           CPA

*         Evaluation of 4000 binucleate cells

 

Table14   Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 20 hrs without S9 mix, Experiment IIB

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	10.0%	-	20 / 40 hrs	87	373	40	1.91	103	341	56	1.91	1.91
Pos. control#^#	100 ng	-	20 / 40 hrs	290	197	13	1.45	296	192	12	1.43	1.44	51.5
Test item	897 µg	-	20 / 40 hrs	111	332	57	1.89	118	344	38	1.84	1.87	4.4
''	1570 µg	-	20 / 40 hrs	154	297	49	1.79	170	306	24	1.71	1.75	17.3
''	2748 µg	-	20 / 40 hrs	153	329	18	1.73	203	285	12	1.62	1.67	25.6

*         c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#             Deion. water

^##        Demecolcine

Table15   Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs with S9 mix, Experiment IIB

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	10.0 %	+	4 / 40 hrs	118	277	105	1.97	121	283	96	1.95	1.96
Pos. control#^#	15.0 µg	+	4 / 40 hrs	270	209	21	1.50	233	230	37	1.61	1.56	42.3
Test item	897 µg	+	4 / 40 hrs	130	295	75	1.89	72	319	109	2.07	1.98	n.c.
''	1570 µg	+	4 / 40 hrs	140	280	80	1.88	153	275	72	1.84	1.86	10.7
''	2748 µg	+	4 / 40 hrs	153	266	81	1.86	117	261	122	2.01	1.93	3.0

*         c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#             Deion. water

^##        CPA

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

 

Table16   Number of micronucleated cells; exposure period 20 hrs without S9 mix, Experiment IIB

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	10.0%	-	20 / 40 hrs	5	0	0	5	8	0	0	8	13	0.65
Pos. control#^#	100 ng	-	20 / 40 hrs	28	0	1	29	14	8	1	23	52	2.60
Test item	897 µg	-	20 / 40 hrs	29	0	0	29	10	0	0	10	39	1.95
''	1570 µg	-	20 / 40 hrs	21	3	1	25	6	0	0	6	31	1.55
''	2748 µg	-	20 / 40 hrs	21	0	1	22	25	0	0	25	47	2.35

^#             Deion. water

^##           Demecolcine

Table17   Number of micronucleated cells; exposure period 4 hrs with S9 mix, Experiment IIB

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	10.0 %	+	4 / 40 hrs	8	1	0	9	12	1	0	13	22	1.10
Pos. control#^#	15.0 µg	+	4 / 40 hrs	45	3	0	48	52	2	1	55	103	5.15
Test item	897 µg	+	4 / 40 hrs	9	0	0	9	9	0	0	9	18	0.90
''	1570 µg	+	4 / 40 hrs	9	2	0	11	13	0	0	13	24	1.20
''	2748 µg	+	4 / 40 hrs	16	2	0	18	14	1	0	15	33	1.65

^#             Deion. water

^##           CPA

Table18   Biometry – Experiment I

Test item versus solvent control [µg/mL]		Preparation interval	Exposure period	S9 mix	Chi^2
Test item	897	40 hrs	4 hrs	-	2.001
"	1570	40 hrs	4 hrs	-	2.001
"	2748	40 hrs	4 hrs	-	1.000
"	897	40 hrs	4 hrs	+	n.c.
"	1570	40 hrs	4 hrs	+	4.512^S
"	2748	40 hrs	4 hrs	+	0.476
Positive control versus solvent control [µg/mL]
MMC	1.2	40 hrs	4 hrs	-	52.685^S
CPA	17.5	40 hrs	4 hrs	+	103.327^S

n.c.  Not calculated as the micronucleus rate is equal or lower than the control rate
^S      Micronucleus rate is statistically significantly higher than the control rate

 

Table19   Biometry – Experiment IIA

Test item versus solvent control [µg/mL]		Preparation interval	Exposure period	S9 mix	Chi^2
Test item	897	40 hrs	20 hrs	-	7.244
"	1570	40 hrs	20 hrs	-	3.492
"	2748	40 hrs	20 hrs	-	0.616
"	897	40 hrs	4 hrs	+	2.143
"	1570	40 hrs	4 hrs	+	6.048^S
"	2748	40 hrs	4 hrs	+	8.817^S
Positive control versus solvent control [per mL]
Demecolcine	150 ng	40 hrs	20 hrs	-	17.565^S
CPA	15.0 µg	40 hrs	4 hrs	+	138.202^S

n.c.  Not calculated as the micronucleus rate is equal or lower than the control rate
^S      Micronucleus rate is statistically significantly higher than the control rate

 

Table20   Biometry – Experiment IIB

Test item versus solvent control [µg/mL]		Preparation interval	Exposure period	S9 mix	Chi^2
Test item	897	40 hrs	20 hrs	-	13.171^S
"	1570	40 hrs	20 hrs	-	7.446^S
"	2748	40 hrs	20 hrs	-	19.560^S
"	897	40 hrs	4 hrs	+	n.c.
"	1570	40 hrs	4 hrs	+	0.088
"	2748	40 hrs	4 hrs	+	2.231
Positive control versus solvent control [per mL]
Demecolcine	100 ng	40 hrs	20 hrs	-	23.787^S
CPA	15.0 µg	40 hrs	4 hrs	+	54.181^S

n.c.  Not calculated as the micronucleus rate is equal or lower than the control rate
^S      Micronucleus rate is statistically significantly higher than the control rate

 

Tables 21 - 24: Historical laboratory control data - Percentage of micronucleated cells in human lymphocyte cultures (2015-2016)

Solvent Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
No. of experiments	78	79
Mean	0.60	0.57
95 % Ctrl limit	0.08 – 1.12	0.12 – 1.03
1x SD	0.26	0.23
Min – Max	0.15 – 1.65	0.05 – 1.35

Solvent Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
No. of experiments	96
Mean	0.62
95 % Ctrl limit	0.16 – 1.08
1x SD	0.23
Min – Max	0.15 – 1.30

Positive Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
	MMC	Demecolcine
No. of experiments	78	81
Mean	12.48	3.72
95 % Ctrl limit	1.44 – 23.52	1.43 – 6.01
1x SD	5.52	1.15
Min – Max	4.15 – 30.30	2.10 – 7.25

Positive Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
	CPA
No. of experiments	165
Mean	5.16
95 % Ctrl limit	0.84 – 9.49
1x SD	2.16
Min – Max	2.10 – 11.90

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore the test substance is considered to be mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.