A mixture of cis- and trans-cyclohexadec-8-en-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the key bacterial reverse mutation assay according to OECD guideline 471, the test item was not mutagenic with and without metabolic activation.

 

In the key Mammalian Cell Gene Mutation Test according to OECD guideline 476, 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-25 to 2021-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

HPRT gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The use of this cell is recommended due to its high proliferation rate, a good cloning efficiency and a stable karyotype with a modal chromosome number of 22.
- Normal cell cycle time: 12 - 16 hours doubling time in stock cultures

For cell lines:
- Absence of Mycoplasma contamination: Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm^2 plastic flasks. About 2-3×10^6 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.
- Doubling time: 12 - 16 hours
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: Yes
- Periodically ‘cleansed’ of spontaneous mutants: Yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
Medium: MEM containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%) for maintainance and seeding (complete culture medium), Complete culture medium without FBS during treatment, Complete culture medium supplemented with 11 μg/mL 6-thioguanine during selection.
CO2 concentration: 1.5 % CO2 (98.5 % air)
Humidity level: Humidified atmosphere
Temperature: 37 °C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/β-naphthoflavone induced rat liver
- Method of preparation of S9 mix: S9 was mixed with MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- Concentration or volume of S9 mix and S9 in the final culture medium: The final protein concentration was 0.75 mg/mL in the cultures.
- Quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

Pre-test for toxicity (with and without S9): 15.9, 31.9, 63.8, 127.6, 255.1, 510.3, 1020.5 and 2041.0 µg/mL

Main experiment (with and without S9): 0.95, 1.9, 3.8, 7.5, 11.3, 15.0, 22.5, 30.0, 45.0 µg/mL. Concentrations of 0.95, 1.9, 3.8, 7.5 µg/mL were used for mutation rate analysis in the experiment without metabolic activation and concentrations of 3.8, 7.5, 11.3, 15.0 and 22.5 µg/mL were used for mutation rate analysis in the experiment with metabolic activation.

Vehicle / solvent:

- Vehicle used: DMSO (test item, positive control with metabolic activation (DMBA)), cell culture medium (positive control without metabolic activation (EMS))

- Justification for choice of solvent/vehicle: Solubility properties, no toxicity and no influence on the outcome of the study at concentrations used.

- Justification for percentage of solvent in the final culture medium:

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

With metabolic activation
DMBA: 2.3 μg/mL = 8.9 μM in DMSO

Without metabolic activation
300 μg/mL = 2.4 mM in cell culture medium

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: One pre-experiment for toxicity and one main experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.7 to 1.2×10^7 cells were seeded for the main experiment. After treatment, at least 2.0×10^6 cells per experimental point were subcultivated. Two additional flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (RS). Three or four days after the first sub-cultivation, at least 2.0×10^6 cells per experimental point were again sub-cultivated. Following the expression time of approximately 7 days, 4 - 5×10^5 cells were seeded in cell culture flasks. Two additional flasks were seeded with approx. 500 cells to determine the viability.
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours (with and without metabolic activation)
- Harvest time after the end of treatment: 8 ± 2 days after the end of treatment (colonies used to determine the relative survival), 9 ± 2 days after the end of the 7-day expression time (colonies used to determine mutation frequency), 8 days after the end of the 7-day expression time (colonies used for evaluation of viability)

FOR GENE MUTATION:
- Expression time: Approx. 7 days
- Selection time: 9 ± 2 days
- Fixation time: 14 - 18 days
- Selective agent used: Cells were exposed to 6-thioguanine (11 µg/mL) for 9 ± 2 days for selection.
- Method to enumerate numbers of viable and mutants cells: Colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency, relative survival (RS)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant colonies per 10^6 cells

Rationale for test conditions:

The concentration range of the main experiment was limited by phase separation and cytotoxicity of the test item. In the main experiment in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. The next higher concentration tested, however, which was separated by a factor smaller than requested by the guideline, could not be evaluated for mutagenicity due to strong toxic effects. In the presence of S9 mix, moderate cytotoxicity was observed at the highest evaluated concentration, which showed phase separation.

Evaluation criteria:

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 on the mean values of culture I and II for the main experiments.

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

A t-test was performed only for the positive controls since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory’s historical negative control data.

However, both, biological and statistical significance will be considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH-value was determined in culture medium of the solvent control (pH 6.99) and of the maximum concentration (pH 6.95) in the pre-experiment without metabolic activation.
- Data on osmolality: The osmolarity was determined in culture medium of the solvent control (471mOsm) and of the maximum concentration (453 mOsm) in the pre-experiment without metabolic activation.
- Precipitation and time of the determination: Phase separation occurred at 15.0 μg/mL and above (without metabolic activation) after four hours treatment. Phase separation occurred at 22.5 μg/mL and above (with metabolic activation) after four hours treatment.

RANGE-FINDING/SCREENING STUDIES: Test item concentrations between 15.9 μg/mL and 2041 μg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. Phase separation occurred at 127.6 μg/mL and above with and without metabolic activation. Cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 63.8 μg/mL and above with and without metabolic activation. There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment.

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

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative survival (RS) and cloning efficiency : See "Attached background material"

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: 0.7 to 1.2×10^7 (number of cells treated), 2.0×10^6 (number of cells subcultivated)
o Number of cells plated in selective and non-selective medium: 4 - 5×10^5 cells
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

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:

This Gene Mutation Assay in Chinese Hamster V79 Cells in vitro (V79/HPRT) according to OECD guideline 476 and GLP was conducted to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (2041 μg/mL) was chosen with respect to the OECD guideline 476 regarding the purity of the test item. The concentration range of the main experiment was limited by phase separation and cytotoxicity of the test item.

In the main experiment, in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. The next higher concentration tested, however, which was separated by a factor smaller than requested by the guideline, could not be evaluated for mutagenicity due to strong toxic effects. In the presence of S9 mix, moderate cytotoxicity was observed at the highest evaluated concentration, which showed phase separation.

No substantial and dose dependent increase of the mutation frequency was observed in the main experiment.

The tested concentrations are shown below:

Exposure period	S9 mix	concentrations in μg/mL
		Main experiment
4 hours	-	0.95	1.9	3.8	7.5	11.3	15.0^PS	22.5^PS	30.0^PS	45.0^PS
4 hours	+	0.95	1.9	3.8	7.5	11.3	15.0	22.5^PS	30.0^PS	45.0^PS

PS = Phase Separation

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-04-02 to 1986-06-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

According to OECD 471, but test with TA102 or E.coli WP2 is lacking

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

yes

Remarks:

Test with TA102 or E.coli WP2 is lacking

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Test with TA102 or E.coli WP2 is lacking

Principles of method if other than guideline:

- Principle of test: According to OECD 471
- Short description of test conditions: According to OECD 471, but the recommended strain for detection of certain oxidising mutagens, cross-linking agents or hydrazines is lacking (E. coli WP2 or TA102)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA1538, TA98) mutations.

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Aroclor 1254 (500 mg/kg bw) induced rats
- Method of preparation of S9 mix: 10% of thawed S9 was mixed with 28 mM MgCl, 33 mM KCl, 5 mM G-6-P, 4 mM NADP and 100 mM (pH 7.4) sodium phosphate
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9 mix (containing 0.05 mL S9) in a final culture medium volume of 2.7 mL
- Quality controls of S9: Metabolic capability was tested with 4-aminobiphenyl and 2-aminoanthracene using S. typhimurium TA 98. A sample of S9 was retained to check for sterility
Concentration of cytochrome P450/ g protein: 0.82 µmol

Test concentrations with justification for top dose:

Pre-experiment I+II for toxicity: 0, 5, 50, 500, 5000, 50000 and 92300 µg/plate (TA 98);
Experiment I: 0, 37, 110, 333, 1,000 and 3,000 µg/plate
Experiment II: 0, 37, 110, 333, 1,000 and 3,000 µg/plate (TA 1535, TA 1538, TA 98) and 0, 4, 12, 37, 111 and 333 µg/plate (TA 1537 and TA 100)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (test substance, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene), water (sodium azide)

- Justification for choice of solvent/vehicle: Solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Remarks:

With S9:
2-AA: 2 µg per 0.1 mL DMSO per plate (all strains)
Without S9:
SA: 1 µg per 0.1 mL DMSO per plate (TA 1535 and TA 100)
2-NF: 2 µg per 0.1 mL DMSO per plate (TA 1538 and TA 98)
9-AA: 80 µg per 0.1 mL DMSO per plate (TA 1537)

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test method: Plate incorporation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Three days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Determination of the number of his+ revertants

Rationale for test conditions:

In the first and second pre-experiment for toxicity, concentrations up to 92300 µg/plate did not reduce the background lawn of TA 98 but precipitation occured at 5000 µg/plate and higher.

In experiment I, the test substance appeared to be slightly toxic for TA 98 at 3,000 µg per plate, for TA 1537 at 1,000 µg per plate (in the absence of the S9 mix) and at 3,000 µg per plate (both in the absence and in the presence of the S9 mix), and for TA 1538 and TA 100 at a dose level of 111.11 µg per plate and above. In addition, a rather strong decrease in the number of his revertants was seen with the strains TA 1537 and TA 100 at a dose level of 333.33 µg/plate and above. Therefore it was decided to carry out the repeat experiment with the strains TA 1537 and TA 100, with dose levels ranging from 4.12 to 333.33 µg/plate.

In experiment II, the test substance was slighly toxic for TA 1538 (both in the absence and in the presence of the S9 mix) at 3000 µg per plate.

Evaluation criteria:

A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies apppearing in the test plates over and above the background spontaneous reversion rate observed with the vehicle, together with evidence of a dose-response.

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated at 5,000 µg/plate and higher. At dose levels of 1,000 and 3,000 µg per plate, a slight precipitate was seen in the plates which did not interfere with the evaluation

RANGE-FINDING/SCREENING STUDIES: In the first and second pre-experiment for toxicity, concentrations up to 92,300 µg/plate did not reduce the background lawn of TA 98 but precipitation occured at 5,000 µg/plate and higher. In view of these observations, 3,000 µg/plate was chosen as the highest dose level for the mutagenicity test.

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

Ames test:
- Signs of toxicity: In experiment I, the test substance appeared to be slightly toxic for TA 98 at 3,000 µg per plate, for TA 1537 at 1,000 µg per plate (in the absence of the S9 mix) and at 3,000 µg per plate (both in the absence and in the presence of the S9 mix), and for TA 1538 and TA 100 at a dose level of 111.11 µg per plate and above. In addition, a rather strong decrease in the number of his revertants was seen with the strains TA 1537 and TA 100 at a dose level of 333.33 µg/plate and above. Therefore it was decided to carry out the repeat experiment with the strains TA 1537 and TA 100, with dose levels ranging from 4.12 to 333.33 µg/plate.

- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

Conclusions:

In a bacterial reverse mutation assay according to OECD guideline 471, the test item was not mutagenic with and without metabolic activation.

Executive summary:

Bacterial Reverse Mutation Test, RL1

The test substance was examined for mutagenic activity in the Ames test according to OECD guideline 471 and GLP, using the histidine requiring Salmonella typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (S9 mix). Concentrations between 0 and 3,000 µg/plate were tested in two independent experiments. The highest tested concentrations were limited by cytotoxicity and/or solubility. It was concluded that the test substance did not show mutagenic activity in any of the strains of S. typhimurium, either in the absence or in the presence of the S9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In the key Mammalian Erythrocyte Micronucleus Test according to OECD guideline 474, the results did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated with 2800 (males) or 4600 mg/kg bw (females) of the test substance.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-06-17 to 1986-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Swiss

Remarks:

CD-1 strain, SPF

Details on species / strain selection:

The mouse is a commonly used species for in vivo genotoxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Not specified
- Weight at study initiation: 33.1-35 g (males), 27.9-29.9 g (females)
- Assigned to test groups randomly: Yes
- Fasting period before study: 2 hours
- Housing: In Makrolon sterilized cages with a grid cover of stainless steel and with a bedding of sterilized softwood chips. Males were housed individually and females five per cage.
- Diet: Ad libitum (except for the 2-hours period before treatment when food was withheld)
- Water: Ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40-60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 0 to day 3

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: None (undiluted test substance), water (negative control), saline (positive control)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was given undiluted by gavage in one single dose of 3 mL/kg bw (corresponding to 2800 mg/kg bw) to males and 5 mL/kg bw (corresponding to 4600 mg/kg bw) to females

Duration of treatment / exposure:

24, 48 and 72 hours

Frequency of treatment:

Single oral dose

Post exposure period:

None

Dose / conc.:

4 600 mg/kg bw (total dose)

Remarks:

Used for females

Dose / conc.:

2 800 mg/kg bw (total dose)

Remarks:

Used for males

No. of animals per sex per dose:

5 males treated with a single dose of 2800 mg/kg bw and 5 females were treated with a single dose of 4600 mg/kg bw. 5 male negative control animals were treated with 3 mL water and 5 female negative control animals were treated with 5 mL water. 2 animals per sex per dose were treated with the positive conrol (1500 mg/kg bw)

Control animals:

yes

Positive control(s):

Mitomycin C
- Justification for choice of positive control: Well-known clastogen, frequently used for in vivo genotoxicity testing
- Route of administration: Intraperitoneally
- Doses / concentrations: 1.5 mg/ kg bw

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: From the results of an acute oral toxicity study, it appeared that the acute oral LD50 of the test substance in mice exceeded 5 mL/kg bw, both in males and in females. Nevertheless the test material induced clear signs of intoxication in male mice. Males treated with 5 mL/kg bw showed tremors, paralysis and piloerection an the first post-treatment day, and 2 out of 5 males died. The female mice showed no signs of intoxication and, deaths did not occur. Therefore, in the present study a lower dose was administered to males than to females, i.e. males were given 3 mL/kg bw and females 5 mL/kg bw.

TREATMENT AND SAMPLING TIMES: At 24 (day 1), 48 (day 2) and 72 (day 3) hours after treatment, 5 males and 5 females of the test group and the negative control group, and 2 males
and 2 females of the positive control group were anaesthetized with ether and killed by decapitation. The femurs were dissected free, the bone marrow was flushed from the femurs into a centiifuge tube containing foetal calf Serum and mixed gently. The cell suspension was centrifuged and the excess serum removed.

DETAILS OF SLIDE PREPARATION: A drop of the Suspension was spread on a slide with a ground glass microscope slide. Ten slides were prepared from each animal. The smears were air dried and fixed in methanol for 5 minutes. The smears were stained with acridine orange for approx. 3 minutes at room temperature and rinsed in the buffer for about 2 minutes. The preparations were mounted with the same buffer and examined on the day of staining with a fluorescence microscope.

METHOD OF ANALYSIS: The incidence of MNC (= micronucleated polychromatic erythrocytes) and the ratio P:N ratio of polychromatic to normochromatic erythrocytes) were scored "blindly" in slides that had been coded, before staining, by a qualified person not involved in the microscopic evaluation of the smears. The incidence of MNC was determined in a total of 1000 polychromatic erythrocytes per animal, by examining 200 polychromatic erythrocytes in each of 5 slides of each animal, for the presence of yellowish-green fluorescing micronuclei. The P:N ratio is used as a measure of cytotoxicity. This ratio was determined by counting the numbers of P and N in a total of 1000 erythrocytes per animal (200 erythrocytes per smear).

Evaluation criteria:

Not described.

Statistics:

The numbers of MNC per 1000 polychromatic erythrocytes and the numbers of polychromatic erythrocytes per 1000 erythrocytes (normo- plus polychromatic erythrocytes) were each analysed by 2 linear logistic regression models with factors: time (24, 48 and 72 hours), sex (male, female) and group, and their mutual interactions. The levels of the factor group were successively: 1) water; test substance and 2) water; mitomycin C.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: No dose range-finding study was conducted. Doses were based on the results of an acute oral toxicity study.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: MNC counts per 1000 polychromatic erythrocytes in water control and test animals were fully comparable (see attached background material).
- Ratio of PCE/NCE: There were no signs of cytotoxic effects of the test compound an the bone marrow cells: the ratios of poly- and normochromatic erythrocytes in control and test animals of the same sex and sacrifice time did not show any indication of treatment related cytotoxic effects attributable to treatment with the test compound.
- Appropriateness of dose levels and route: The substance was tested in concentrations which induced slight toxicity but no severe suffering or deaths. The oral route was chosen based on the most probable route of exposure.
- Clinical signs: Piloerection occured 4 hours after treatment in all animals. Three animals (2 males and 1 female) showed paralysis of the hind legs. 24 hours after treatment, one male animal died and some other animals showed rough coats. Males appeared more affected by treatment than females. No specific signs of toxicity were observed at 48 and 72 hours after treatment.

Conclusions:

In a mammalian erythrocyte micronucleus test according to OECD guideline 474, the results did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated with 2800 (males) or 4600 mg/kg bw (females) of the test substance.

Executive summary:

In an in vivo mammalian erythrocyte micronucleus test according to OECD guideline 474 and GLP, the test item was examined using mice as experimental animals. Two groups of 15 males and 15 females each were treated once orally with, respectively, 3 mL (2.8 g) and 5 mL (4.6 g) of the undiluted test substance per kg body weight. Negative control animals were treated in a comparable way with water only. A positive control group of 6 males and 6 females was treated once intraperitoneally with 1.5 mg of the mutagen Mitomycin C per kg body weight. At 24, 48 and 72 hours after treatment, ten negative controls (5/sex), ten test animals (5/sex) and four positive controls (2/sex) were killed. Samples of bone marrow were collected and bone marrow smears were prepared for microscopic examination. Treatment with- the test material induced overt signs of intoxication and one male died. Mean body weights were not affected. The incidence of micronucleated erythrocytes and the ratios of poly- to normochromatic erythrocytes in test animals were fully comparable to those found in the concomitant negative controls killed at the same exposure time. Animals treated with the mutagen mitomycin C showed an increased incidence of micronucleated polychromatic erythrocytes and a decrease in the ratio of poly— to normochromatic erythrocytes. It was concluded that the results of the micronucleus test did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated with a high dose of the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Key, Bacterial Reverse Mutation Test, RL1

The test substance was examined for mutagenic activity in the Ames test according to OECD guideline 471 and GLP, using the histidine requiring Salmonella typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and a liver microsome fraction of Aroclor-induced rats for metabolic activation (S9 mix). Concentrations between 0 and 3,000 µg/plate were tested in two independent experiments. The highest tested concentrations were limited by cytotoxicity and/or solubility. It was concluded that the test substance did not show mutagenic activity in any of the strains of S. typhimurium, either in the absence or in the presence of the S9 mix.

 

Key, Mammalian Cell Gene Mutation Test in vitro, RL1

This Gene Mutation Assay in Chinese Hamster V79 Cells in vitro (V79/HPRT) according to OECD guideline 476 and GLP was conducted to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (2041 μg/mL) was chosen with respect to the OECD guideline 476 regarding the purity of the test item. The concentration range of the main experiment was limited by phase separation and cytotoxicity of the test item.

In the main experiment, in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. The next higher concentration tested, however, which was separated by a factor smaller than requested by the guideline, could not be evaluated for mutagenicity due to strong toxic effects. In the presence of S9 mix, moderate cytotoxicity was observed at the highest evaluated concentration, which showed phase separation.

No substantial and dose dependent increase of the mutation frequency was observed in the main experiment.

The tested concentrations are shown below:

Exposure period	S9 mix	concentrations in μg/mL
		Main experiment
4 hours	-	0.95	1.9	3.8	7.5	11.3	15.0^PS	22.5^PS	30.0^PS	45.0^PS
4 hours	+	0.95	1.9	3.8	7.5	11.3	15.0	22.5^PS	30.0^PS	45.0^PS

PS = Phase Separation

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.

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.

 

Key, In vivo Mammalian Erythrocyte Micronucleus Test, mice, RL1

In an in vivo mammalian erythrocyte micronucleus test according to OECD guideline 474 and GLP, the test item was examined using mice as experimental animals. Two groups of 15 males and 15 females each were treated once orally with, respectively, 3 mL (2800 mg) and 5 mL (4600 mg) of the undiluted test substance per kg body weight. Negative control animals were treated in a comparable way with water only. A positive control group of 6 males and 6 females was treated once intraperitoneally with 1.5 mg of the mutagen Mitomycin C per kg body weight. At 24, 48 and 72 hours after treatment, ten negative controls (5/sex), ten test animals (5/sex) and four positive controls (2/sex) were killed. Samples of bone marrow were collected and bone marrow smears were prepared for microscopic examination. Treatment with- the test material induced overt signs of intoxication and one male died. Mean body weights were not affected. The incidence of micronucleated erythrocytes and the ratios of poly- to normochromatic erythrocytes in test animals were fully comparable to those found in the concomitant negative controls killed at the same exposure time. Animals treated with the mutagen mitomycin C showed an increased incidence of micronucleated polychromatic erythrocytes and a decrease in the ratio of poly— to normochromatic erythrocytes. It was concluded that the results of the micronucleus test did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated with a high dose of the test substance.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified and labelled according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.