Calcium bis[4-[[1-[[(2-methylphenyl)amino]carbonyl]-2-oxopropyl]azo]-3-nitrobenzenesulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: negative, according to OECD TG 471, GLP-compliant, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without metabolic activation, 2020, K1

HPRT: negative, according to OECD TG 476, GLP-compliant, V79 cell line, with and without metabolic activation, 2020, K1
Micronucleus test: negative, according to OECD TG 487, GLP-compliant, Human lymphocytes, with and without metabolic activation, 2020, K1

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 JUN 2020 - 27 AUG 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 98.6 area-%
Content: 90.7 (± 0.2) g/100 g
Water content: 4.0 g/100 g
Identity: confirmed
Date of production: 05 May 2011
Physical state, appearance: solid, yellow
Storage conditions: room temperature

Target gene:

his, trp

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9 fraction
Uninduced hamster liver S9 fraction

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2800 and 5600 μg/plate

3 test plates per dose or per control
1st Experiment: Standard plate test with and without S9 mix (SPT)
2nd Experiment: Preincubation test with and without S9 mix (PIT)
3rd Experiment: Prival preincubation test with and without S9 mix (Prival)

Due to the chemical structure of the test substance and on request of the sponsor the Amestest with prival modification was neccessary.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the limited solubility 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:

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

Remarks:

Without S9 mix

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:

Without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix

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:

Without S9 mix

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:

With hamster liver S9 mix; all strains

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: congo red

Remarks:

With hamster liver S9 mix; TA 98

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:

With rat liver S9 mix; all strains

Details on test system and experimental conditions:

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


DURATION
- Standard plate test:
After mixing, the samples were poured onto Minimal glucose agar plates within approx. 30 seconds.

- Preincubation Test:
Preincubation at 37°C for the duration of about 20 minutes using a shaker.

- Prival Preincubation Test:
Preincubation at 37°C for the duration of about 30 minutes using a shaker.

- Exposure duration (all experiments): 48 – 72 hours


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


STERILITY CONTROL
- Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains

Evaluation criteria:

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

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

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was observed at and above 333 μg/plate both with and without S9 mix.
- A bacteriotoxic effect (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 100 μg/plate.
- In the preincubation assay bacteriotoxicity (decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at and above 1000 μg/plate.
- In the prival preincubation assay bacteriotoxicity (decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at and above 333 μg/plate.

Conclusions:

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

Executive summary:

The test article was tested in the Ames reverse mutation assay according to OECD guideline 471 and in compliance with GLP, using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA. Three experiments were performed with doses ranging from 33 to 5600 µg/plate (vehicle: DMSO): A standard plate test (SPT), preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) and prival preincubation test (Prival) with and without metabolic activation (liver S9 mix from uninduced hamsters). Precipitation of the test substance was observed at and above 333 μg/plate both with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions at and above 100 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, in the preincubation test or prival preincubation test without S9 mix or after the addition of a metabolizing system.

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

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 JUN 2020 - 01 SEP 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline Kanpoan No. 287

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

Purity: 98.6 area-%
Content: 90.7 (± 0.2) g/100 g
Identity: confirmed
Homogeneity: Given
Expiry date: 05 May 2021
Physical state, appearance: solid, yellow
Storage conditions: room temperature
Stability in Solvent: Not determined

The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany)
- Suitability of cells: recommended cell line due to a high proliferation rate and a good cloning efficiency of untreated cells

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: not specified
- Methods for maintenance in cell culture: 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.
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- 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 treatment no FBS was added to the medium. 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).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. 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. The S9 fraction will be used at concentrations of approx. 2% v/v in final test medium. 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 31.7 mg/mL in the pre-experiments and in the main experiment.

Test concentrations with justification for top dose:

0.9, 1.9, 3.8, 7.5, 15.0, 30.0, 60.0 µg/mL

The dose range of the main experiment was set according to data generated in the pre-experiment.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.
The cultures at the three highest concentrations with and without metabolic activation were not continued to avoid analysis of too many precipitating concentrations.

Vehicle / solvent:

Vehicle: suspension in culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: approximately 0.7 to 1.2×10^7 cells were seeded in plastic flasks
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: no
- Exposure duration/duration of treatment: 4 h
- Harvest time after the end of treatment: immediately after end of treatment

FOR GENE MUTATION:
- Expression time: 8 days (evaluation for viability) and 11 days (mutation analysis)
- Fixation time: 8 d after treatment
- Colonies with more than 50 cells were counted.

Evaluation criteria:

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 10^6 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 statistically 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.

Interpretation 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 (e.g. Poisson-based 95% control limits).

Statistics:

The statistical analysis was performed in the mean values of culture I and II for all experiments.
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mean mutant frequencies. The mean number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

However, both, biological and statistical significance were considered together.

A t-Test was not performed since all mean mutant frequencies were well within the 95% confidence interval of our laboratory’s historical negative control data.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The mean mutant frequency obtained in the main experiment for the solvent control was 11.6 mutants per 10^6 cells without metabolic activation, and 14.3 mutants per 10^6 cells with metabolic activation. The values were well within the 95% confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476.
The range of the mutant frequencies of the groups treated with the test item was from 10.6 up to 17.6 mutants per 10^6 cells.
Referring to the mean values no statistical significant linear regression was observed. Furthermore no increase in the number of mutant colonies (mean values) above the 95% confidence interval was observed.
EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Table 1: Summary of results

				relative	relative	rel. adjusted	mutant	95%
	conc.	P	S9	cloning	cell	cloning	colonies/	confidence
	µg/mL		mix	efficiency I	density	efficiency I	10^6 cells	interval
				%	%	%
Column	1	2	3	4	5	6	7	8
Main Experiment / 4 h treatment				mean values of culture I and II
Solvent control with medium			-	100.0	100.0	100.0	11.6	3.5 -31.0
Positive control (EMS)	300.0		-	92.8	94.7	87.5	224.6	3.5 -31.0
Test item	0.9		-	104.1	80.9	84.4	11.4	3.5 -31.0
Test item	1.9		-	104.0	90.9	93.6	10.9	3.5 -31.0
Test item	3.8		-	101.3	106.1	106.5	17.6	3.5 -31.0
Test item	7.5	P	-	100.3	88.4	87.9	11.7	3.5 -31.0
Test item	15.0	P	-	culture was not continued#
Test item	30.0	P	-	culture was not continued#
Test item	60.0	P	-	culture was not continued#
Solvent control with medium			+	100.0	100.0	100.0	14.3	4.2 -30.7
Positive control (DMBA)	2.3		+	102.7	93.3	95.2	93.4	4.2 -30.7
Test item	0.9		+	102.6	108.8	111.5	11.2	4.2 -30.7
Test item	1.9		+	101.3	97.2	97.3	14.6	4.2 -30.7
Test item	3.8		+	99.5	101.5	101.0	10.6	4.2 -30.7
Test item	7.5	P	+	98.5	96.6	95.0	11.7	4.2 -30.7
Test item	15.0	P	+	culture was not continued#
Test item	30.0	P	+	culture was not continued#
Test item	60.0	P	+	culture was not continued#

P = precipitationat at the end of treatment

# culture was not continued to avoid analysis of too many precipitating concentrations

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

Executive summary:

The study according to OECD TG 476 (GLP compliant) was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in one main experiment using two parallel cultures. The experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The maximum test item concentration in the pre-experiment was 250.0 μg/mL based on the solubility properties of the test item. The maximum concentration in the main experiment was 60.0 μg/mL based on precipitation observed in the pre-experiment. The test item was suspended in cell culture medium.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment.
Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 JUL 2020 - 18 SEP 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Purity: 98.6 area-%
Content: 90.7 (± 0.2) g/100 g
Identity: confirmed
Homogeneity: Given
Expiry date: 05 May 2021
Physical state, appearance: solid, yellow
Storage conditions: room temperature
Stability in Solvent: Not determined

The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test Item Preparation:
Stock formulations of the test item (stirred for at least one hour) and serial dilutions were made in culture medium. All formulations were prepared freshly before treatment and used within two hours of preparation.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human lymphocytes
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.

For lymphocytes:
- Sex, age and number of blood donors:
- Experiment I: One female (20 years old), healthy, non-smoking
- Experiment II: One male (19 years old), healthy, non-smoking
- Whether blood from different donors were pooled or not: no pooling
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
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).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the 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 used for this study was 31.7 mg/mL (Lot no. 200220).

Test concentrations with justification for top dose:

Experiment I (4h exposure): 1.6, 2.8, 5.0, 8.7, 15.2 (P), 26.7 (P), 46.6 (P), 81.6 (P), 143 (P), 250 µg/mL (P) (with and without S9 mix)
Experiment II (20 h exposure): 19.9, 34.8, 60.9 (P), 107 (P), 87 (P), 327 (P), 571 (P), 1000 µg/mL (P) (without S9 mix)

(P): Precipitation was observed by the unaided eye at the end of treatment

A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment.

Vehicle / solvent:

Cell culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Demecolcine

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: Continous exposure (without S9 mix); Pulse exposure (with/without S9 mix)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: no preincubation
- Exposure duration/duration of treatment: 4 h and 20 h
- Harvest time after the end of treatment: 16 h recovery (only 4 h exposure) + 20 h CytB exposure (all cultures)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytochalasin B (4 μg/mL) was added and the cells were cultured approximately 20 hours until preparation
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): fixation with methanol and glacial acetic acid (19 parts plus 1 part, respectively), slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide, the cells were stained with Giemsa, mounted after drying and covered with a coverslip.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

CBPI= (MONC x 1) + (BINC x 2) + (MUNC x3) / n

CBPI Cytokinesis-block proliferation index
n Total number of cells
MONC Mononucleate cells
BINC Binucleate cells
MUNC Multinucleate cells

Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]

T Test item
C Solvent control

Evaluation criteria:

Acceptability Criteria

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

Interpretation of Results

A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval).

A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval).

Statistics:

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

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Solvent control: pH = 7.61; Test item: pH = 7.43
- No relevant influence on osmolarity or pH was observed.
- In Experiment I, precipitation of the test item in the culture medium was observed at 15.2 μg/mL and above in the absence and presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 60.9 μg/mL and above at the end of treatment.

RANGE-FINDING/SCREENING STUDIES:
- A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

STUDY RESULTS
- Demecolcine (75 ng/mL), MMC (0.8 μg/mL) or CPA (15.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei. The value of the positive control Demecolcine exceeded the range of the historical control data. As this observation confirms the response of the test system, it does not affect the validity of the experiment.

HISTORICAL CONTROL DATA:
- The historical control data were generated in accordance with the OECD Guideline 487.
For the solvent controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The calculated 95% control limit of the solvent controls (realised as 95% confidence interval) was applied for the evaluation of acceptability and interpretation of the data. Control charts of the corresponding experiments are added as quality control method.
For the positive controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The min-max range of the positive controls was applied for the evaluation of acceptability. Control charts of the corresponding experiments are added as quality control method.

Table 2: Summary of results

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells	95% Ctrl limit
		in µg/mL	CBPI		in %**	in %
Exposure period 4 h without S9 mix
I	40 h	Solvent control^1	1.80		0.55	0.00 – 1.04
		Positive control^2	1.62	22.8	9.00^S
		5.0	1.83	n.c.	0.85
		8.7	1.77	3.1	0.55
		15.2^P	1.78	2.4	0.65
Trend test: p-value 0.952
Exposure period 20 h without S9 mix
II	40 h	Solvent control^1	1.61		0.45	0.00 – 0.86
		Positive control^3	1.31	49.3	8.30^S
		19.9	1.62	n.c.	0.40
		34.8	1.50	17.9	0.10
		60.9^P	1.50	19.1	0.75
Trend test: p-value 0.613
Exposure period 4 h with S9 mix
I	40 h	Solvent control^1	1.62		0.70	0.00 – 1.03
		Positive control^4	1.33	46.7	5.15^S
		5.0	1.63	n.c.	0.70
		8.7	1.66	n.c.	0.75
		15.2^P	1.66	n.c.	0.35
Trend test: p-value 0.234

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

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

P Precipitation occurred at the end of treatment

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

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

1 Culture medium

2 MMC 0.8 μg/mL

3 Demecolcine 75 ng/mL

4 CPA 15.0 μg/mL

Conclusions:

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

Executive summary:

In this study according to OECD TG 487 and with GLP-compliance, the test item, suspended in cell culture medium, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure periods were 4 hours with and without S9 mix. In Experiment II, the exposure period was 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. 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, 1000 μg/mL, was chosen with regard to the solubility properties and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

In Experiment I, precipitation of the test item in the culture medium was observed at 15.2 μg/mL and above in the absence and presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 60.9 μg/mL and above at the end of treatment.

No relevant influence on osmolarity or pH was observed.

In both experiments in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In both experiments in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genotoxicity in vitro:

The test article was tested in the Ames reverse mutation assay according to OECD guideline 471 and in compliance with GLP, using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA. Three experiments were performed with doses ranging from 33 to 5600 µg/plate (vehicle: DMSO): A standard plate test (SPT), preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) and prival preincubation test (Prival) with and without metabolic activation (liver S9 mix from uninduced hamsters). Precipitation of the test substance was observed at and above 333 μg/plate both with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions at and above 100 μg/plate. No relevant increase in the number of his+ or trp+ revertants could be observed in the three different tests in presence and absence of a metabolizing system.

 

In a further study according to OECD TG 487 and with GLP-compliance, the test item, suspended in cell culture medium, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation (S9 mix).
Two independent experiments were performed. In Experiment I, the exposure periods were 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. In each experimental group, two parallel cultures were analyzed. 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, 1000 μg/mL, was chosen with regard to the solubility properties and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

In both experiments in the absence and presence of S9 mix, no cytotoxicity and no relevant increase in the numbers of micronucleated cells were observed after treatment with the test item.

 

The third in vitro study according to OECD TG 476 (GLP compliant) was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in one main experiment using two parallel cultures. The experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The maximum concentration in the main experiment was 60.0 μg/mL based on precipitation observed in the pre-experiment. The test item was suspended in cell culture medium. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in vitro micronucleus assay (OECD 487, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Regulation (EC) No. 2020/1182.