1,1'-[oxybis(ethyleneoxy)]diethylene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in a bacterial reverse mutation assay in Salmonella and E.coli (OECD 471, Ames). Also, no mutagenic effects were observed in an in vitro chromosome aberration test using primary human blood lymphocytes and in an in vivo mouse micronucleus test after oral administration (OECD 473, CA). In addition, the substance showed no mutagenic effects in a gene mutation test in vitro (OECD 476, HPRT).

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:

1996-04 to 1996-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

92/69/EEC

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction from livers of Sprague-Dawley rats receiving a single intraperitoneal injection of Aroclor 1254

Test concentrations with justification for top dose:

1st and 2nd experiment: 0, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 Mix, E.coli

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 Mix, TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Mix, TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

without S9 Mix, TA100 ,TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

with S9 Mix, all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 - 72 h at 37°C
- Number of independent experiments: 2
- Number of replicates: 3 plates per concentration

DETERMINATION OF CYTOTOXICITY
- Method: background growth, decrease in the number of revertants

Evaluation criteria:

In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found.

A weakly bacteriotoxic effect was observed with metabolic activation in the preincubation test at doses >= 2500 µg/plate (TA 1535, TA 1537, TA 98, E. coli).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-01-08 to 2007-02-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking 30 year old adult female on 08 January 2007 for the preliminary toxicity assay and from the same donor on 23 January 2007 for the definitive assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9

Test concentrations with justification for top dose:

Preliminary Toxicity Assay
0.158, 0.474, 1.58, 4.74, 15.8, 47.4, 158, 474, 1580 µg/mL

Chromosome Aberration Assay
125, 250, 500, 1000, 1580 µg/mL (125, 250 µg/mL were not scored)

Vehicle / solvent:

- Solvent used: Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Mix

Details on test system and experimental conditions:

TREATMENT OF CELLS WITH TEST SUBSTANCE:
Two independent experiments were performed. Duplicate cultures were used for each treatment.

Exposure period:
- with metabolic activation: first and second test: 4 hours
- without metabolic activation: first test: 4 hours; second test: 20 hours

Harvest times for all tests were 20 hours. Two hours before the cells were harvested mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 0.1 µg/mL.

MICROSCOPIC EXAMINATION:
The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted. Metaphase cells with 46 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate treatment condition) were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphase spreads that were examined and scored per duplicate flask was reduced when the percentage of aberrant cells reached a significant level (at least 10%) before 100 cells are scored.

DETERMINATION OF CYTOTOXICITY
- Method: Cells were collected by centrifugation, treated with hypotonic potassium chloride, fixed, stained and the number of cells in mitosis per 500 cells scored was determined in order to evaluate the effect on mitotic index.

OTHER EXAMINATIONS:
- Determination of polyploidy and endoreplication: The percent polyploid and endoreduplicated cells was evaluated per 100 cells.

Evaluation criteria:

The toxic effects of the treatment are based upon mitotic inhibition relative to the solvent-treated control and are presented for the preliminary toxicity test and the chromosome aberration assay. The number and types of aberrations per cell, the percentage of structurally and numerically damaged cells (percent aberrant cells), and the frequency of structural aberrations per cell (mean aberrations per cell) in the total population of cells examined was calculated and reported for each treatment group. Chromatid and isochromatid gaps were also presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's Exact test. Fisher's Exact test was used to compare pair wise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's Exact test at any test item dose level, the Cochran-Armitage test was used to measure dose-responsiveness.
All conclusions were based on sound scientific judgment; however, as a guide to interpretation of the data, the test artic1e was considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner with one or more concentrations being statistically elevated relative to the solvent control group (p<0.05). However, values that are statistically significant but do not exceed the range of historical solvent controls may be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

Key result

Species / strain:

lymphocytes: Peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at least 50% reduction in mitotic index relative to the solvent control

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Solubility Test
Water was the solvent of choice based on the solubility of DEGDVE and compatibility with the target cells. The test article formed a workable suspension in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.

Dose Formulation Analysis
The results of the analysis for the analyzed samples (top, middle and bottom of 15.8 mg/mL concentration) did not meet the acceptance criterion of 80-120% of target concentration. The actual analytical results found that the analyzed dose level contained an average of 73.6% of target concentration. The analytical assay acceptance criterion of < 10% relative standard deviation (RSD) was met and the formulation was determined to be homogeneous. No test article was detected in the vehicle control.
Although the analyzed sample was not within ±20% of target, the toxicity observed (at least 50% reduction in mitotic index relative to the solvent control) at the high dose of 1580 µg/mL in the non-activated 20-hour exposure group confirms that the maximum dose that the test system could tolerate was achieved. Therefore, the analytical results for the dosing solutions and the observed deviation from nominal concentration did not adversely impact the integrity of the data or the validity of the study conclusion.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-11-23 to 2021-03-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

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:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO (Chinese hamster ovary) cell line
- Suitability of cells: proven

For cell lines:
- Absence of Mycoplasma contamination: Each batch used for mutagenicity testing was checked for mycoplasma contamination
- Methods for maintenance in cell culture:
For cell cultivation, deep-frozen cell suspensions were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL Ham's F12 medium including 10% (v/v) FCS. Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm2 plastic flasks).
- Cell cycle length, doubling time or proliferation index:
high proliferation rate (doubling time of about 12 - 16 hours)
high plating efficiency (about 90%)
- Modal number of chromosomes: karyotype with a modal number of 20 chromosomes
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS). Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: rat liver

- method of preparation of S9 mix: The S9 fraction was prepared according to Ames et al. 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)] received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally 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 45 - 65%. The day/night rhythm was 12 hours: light from 6 am – 6 pm and darkness from 6 pm – 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

- concentration or volume of S9 mix and S9 in the final culture medium: The S9 mix was prepared freshly prior to each experiment. A sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and main experiments. This preparation, the S9 mix (10% S9 fraction), was kept on ice until used.

Test concentrations with justification for top dose:

1st Exp: 50, 100, 200, 400, 800 and 1600 µg/mL (with and without S9 mix, 4-hour exposure)
2nd Exp.:100, 200, 400, 800,1200 and 1600 µg/mL (with and without S9 mix, 4-hour exposure)

Vehicle / solvent:

- Vehicle used: DMSO

- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in culture medium and in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available.

- Justification for percentage of solvent in the final culture medium: The final concentration of the vehicle DMSO in culture medium will be 1% (v/v) and is well tolerated by the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

400 µg/mL, without S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

1.25 µg/mL, with S9 Mix

Details on test system and experimental conditions:

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

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

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

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 - 9 days
- Selection time: 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16
- Selective agent is used: 6-thioguanine (10 μg/mL) for one week
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
For selection of the mutants, two 175 cm2 flasks with 2x10E6 cells each from every treatment group, were seeded in 20 mL selection medium ("TG" medium) at the end of the expression period. Only the cells resistant to 6-thioguanine that were assumed to be deficient of HPRT survived. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted. The number of colonies in each flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10E6 cells seeded.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
pH: The pH was measured at least for the top concentrations and for the vehicle controls with and without S9 mix at the beginning of test substance treatment.
Osmolality: Osmolality was measured in at least the top concentrations and the vehicle controls with and without S9 mix at the beginning of test substance treatment.
Solubility: Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.

Rationale for test conditions:

Following the requirements of the current international guidelines and the ICPEMC Task Group a test substance should be tested up to a maximum concentration of 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top dose should result in approximately 10 - 20% relative survival (adjusted cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of the exposure period.

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
-A statistically significant increase in mutant frequencies is obtained.
-A dose-related increase in mutant frequencies is observed.
-The corrected mutation frequencies (MFcorr.) exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit)

Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
-Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
-The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit)

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose. The calculation was performed using EXCEL function RGP. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction. However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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: pH values were not relevantly influenced by test substance treatment
- Data on osmolality: Osmolality was not relevantly influenced by test substance treatment
- Precipitation and time of the determination: In the main study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest required test substance concentration.

After 4 hours treatment neither in the absence nor presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test group tested for gene mutations

RANGE-FINDING/SCREENING STUDIES
In the pre-test for toxicity based on the purity and the molecular weight the substance was used as top concentration both with and without S9 mix at 4 hour exposure time. The pre-test was performed following the method described for the main experiment. The relative survival (RS) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. In the pre-test the pH value and osmolality were not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.

Precipitation of the test substance in the vehicle dimethyl sulfoxide (DMSO) was not observed in the stock solution (Test group: 160 mg/mL). In culture medium, test substance precipitation occurred in the presence of S9 mix from 800.0 µg/mL onward. In the absence of S9 mix no test substance precipitation was observed. After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20%.

STUDY RESULTS

- Results from cytotoxicity measurements: In both experiments, there was no decrease in the number of colonies as described by the relative survival in the presence and absence of S9 mix up to the highest evaluated concentrations for gene mutation.

- Genotoxicity results:
In the 1st Experiment in the absence of metabolic activation, the values for the corrected mutation frequencies (MFcorr.) ranged between 0.40 – 6.90 per 10E6 cells, the respective vehicle control value had 5.40 per 10E6 cells The values in the test groups 800.0 µg/mL (MFcorr.: 6.64 per 10E6 cells), and 1600.0 µg/mL (MFcorr.: 6.90 per 10E6 cells) were slightly above the range of the 95% control limit (without S9 mix: MFcorr.: 0.00 – 6.21 per 10E6 cells). Nevertheless, the values were not statistically significant. Furthermore, a concentration related increase in the mutant frequencies was also not observed. The relevance of the observation in the 1st Experiment was assessed in a 2nd Experiment. In the 2nd Experiment in the absence of metabolic activation, the values for the corrected mutation frequencies ranged between MFcorr.: 0.32 -3.28 per 10E6 cells; the respective vehicle control value had 1.10 per 10E6 cells. The obtained values were within the range of the 95% vehicle control limit (without S9 mix: MFcorr.: 0.00 – 6.21 per 10E6 cells). A concentration related increase in the mutant frequencies was not observed and no statistically significant increase in the mutant frequencies was determined In the 1st Experiment in the presence of metabolic activation, the values for the corrected mutation frequencies (MFcorr.) ranged between 5.17 – 7.95 per 106 cells, the respective vehicle control value had 5.98 per 10E6 cells. The value in the test group 200.0 µg/mL (MFcorr.: 7.95 per 10E6 cells) was slightly above the range of the 95% control limit (with S9 mix: MFcorr.: 0.00 – 7.08 per 10E6 cells). The value was, however, not statistically significant as compared to the vehicle control value. Furthermore, a concentration related increase in the mutant frequencies was also not observed. The relevance of the observation in the 1st Experiment was assessed in a 2nd Experiment.
In the 2nd Experiment in the presence of metabolic activation, the values for the corrected mutation frequencies ranged between MFcorr.: 0.74 -6.16 per 10E6 cells; the respective vehicle control value had 1.70 per 106 cells. The value in test group 800.0 µg/mL (MFcorr.: 6.16 per 10E6 cells) was statistically significantly increased compared to the concurrent vehicle control value, nevertheless within the range of the 95% vehicle control limit (with S9 mix: MFcorr.: 0.00 – 7.08 per 10E6 cells). A concentration related increase in the mutant frequencies was not observed. The positive control substances EMS (without S9 mix; 400 µg/mL) and DMBA (with S9 mix; 1.25 µg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 49.41 – 84.64 per 10E6 cells; with S9 mix: MFcorr.: 33.06 – 129.91 per 10E6 cells) were within our historical positive control data range (without S9 mix: MFcorr.: 42.47 – 438.29 per 10E6 cells; with S9 mix: MFcorr.: 21.52 – 270.48 per 10E6 cells.

The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

The mutation frequencies of the vehicle control groups were within the historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data.

HISTORICAL CONTROL DATA
See attached tables.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance was not clastogenic in a study according to OECD 474 in the mouse micronucleus assay.

 

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:

2006-12-21 to 2007-01-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, MD, USA
- Age at study initiation: Approximately 6 to 8 weeks
- Weight at study initiation:
Pilot Toxicity Study: Males: 26.0-28.7 g Females: 22.9-24.8 g
Definitive Micronucleus Study: Males: 25.5-30.1 g Females: 22.8-28.0 g
- Assigned to test groups: The mice were assigned to seven experimental groups of five males and five females each according to a computer-generated
program, which is based on distribution according to body weight.
- Housing: Mice of the same sex were housed up to five per rodent Micro-Barrier cage.
- Diet: Ad libitum. Certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet)
- Water: Ad libitum
- Acclimation period: Not less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F
- Humidity: 50 ± 20%
- Air changes: Micro-VENT full ventilation, HEPA filtered system
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Corn oil
- Justification for choice of solvent/vehicle: Corn oil was chosen as the most appropriate vehicle for the test article vehicle based on test item solubility and compatibility of the vehicle with the test system. The test article was soluble in corn oil at 100 mg/mL, the maximum concentration tested in the study.
- Concentration of test material in vehicle: All dose formulations were administered by oral gavage at a dose volume of 20 mL/kg.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article dose formulations were prepared fresh for each phase of the study, just prior to dose administration. Each time, the formulations were prepared by combining an appropriate amount of test article with an appropriate amount of vehicle following by vortexing. The formulations were not adjusted for purity of the test article. All formulations appeared to be yellow solutions.

Duration of treatment / exposure:

Single oral dose

Frequency of treatment:

Single oral dose

Post exposure period:

24 h (0, 500, 1000, 2000 mg/kg bw) and 28 h (0, 2000 mg/kg bw) post-dose

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Low dose (500 mg/kg): 5 animals
Mid dose (1000 mg/kg) 5 animals
High dose (2000 mg/kg): 10 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

-Positive control: Cyclophosphamide monohydrate (CP, CAS number 6055-19-2)
- Justification for choice of positive control(s):
- Route of administration: Oral gavage
- Doses / concentrations: 50 mg/kg; 2.5 mg/mL

Tissues and cell types examined:

Bone marrow was collected and bone marrow smears (slides) were prepared. Bone marrow cells (polychromatic erythrocytes) were examined microscopically for the presence of micronuclei.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Selection of doses for the definitive micronucleus assay were based on the toxicity of the test article.

TREATMENT AND SAMPLING TIMES:
Groups of mice were exposed to a single oral dose at either 500, 1000 or 2000 mg/kg, or the vehicle control or the positive control article. All dose formulations were administered by oral gavage at a dose volume of 20 mL/kg. All mice in the experimental and control groups were weighed immediately before dose administration and the administered volume was based on individual body weight. Mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity.

DETAILS OF SLIDE PREPARATION:
At the scheduled bone marrow collection time, five mice per sex per treatment were euthanized by CO2 asphyxiation. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:

Using a light microscope and a medium magnification (400X), an area of acceptable quality was selected such that the cells were well spread and stained.
Using oil immersion (1000X), the following cell populations were evaluated and enumerated:
• Polychromatic erythrocytes (PCEs)
PCEs stain bluish. Two-thousand PCEs per mouse were screened (scored) for the presence of micronuclei resulting in evaluation of a total of 10,000 PCEs per each treatment group.
• Normochromatic erythrocytes (NCEs)
NCEs stain pink (redish). The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) is determined for each animal in order to determine the proportion of polychromatic erythrocytes to total erythrocytes(pCEs/ECs). The incidence of MNCEs per 2000 PCEs was enumerated for each animal.
• Micronuc1ei (M)
Micronuclei are round, darkly-staining nuc1ear (chromosome) fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs). The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes per each animal (pCEs/ECs ratio).

Evaluation criteria:

The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10,000 PCEs for each treatment group was determined. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each mouse and treatment group (PCEs/ECs ratio). The proportion of polychromatic erythrocytes to total erythrocytes in test article-treated animals should not be less than 20% of the control value.
All conclusions were based on a scientific judgment. As a guide to interpretation of the data, the following was considered:
• The test article was considered to induce a positive response if a dose-responsive increase in the incidence of micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p: < 0.05, Kastenbaum-Bowman Tables) at any sampling time.
• Values that were statistically significant but did not exceed the range of historical negative controls were judged as not biologically significant/relevant.
• The test article was judged negative if no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.

Statistics:

Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution . All analyses were performed separately for each sex and sampling time.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility Test
Corn oil was chosen as the most appropriate vehicle for the test article vehicle based on DEGDVE solubility and compatibility of the vehicle with the test system. The test article was soluble in corn oil at 100 mg/mL, the maximum concentration tested in the study.

Definitive Micronucleus Study
No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs noted within two hours after dosing included: lethargy in 3/10 high dose (2000 mg/kg) males and prostration in 1/10 high dose males. Piloerection also occurred within 2 hours of dose administration in all high dose (2000 mg/kg) animals in both sexes and again was noted in up to 3/10 high dose animals in each sex on the days following dose administration. All other mice treated with test and control articles appeared normal throughout the study.

Dose Formulation Analysis
Samples of dose formulations used in the definitive study at 0.0 mg/mL (vehicle), 25, 50 and 100 mg/mL were collected and measured.
Based on the results, concentrations of 50 and 100 mg/mL were 92.3% and 92% of target, respectively. These results indicate that the mid and highest concentrations met the acceptance criteria of ± 15 % of target and <5% RSD (relative standard deviation). The first analysis of 25 mg/mL (low concentration) failed to meet the acceptance criteria, but the repeat analysis confirmed that this concentration was within the acceptable range of target (99.4%), but the % RSD was outside of the acceptable range of <5% (6.28%). This out of target result for the low concentration did not adversely impact the outcome of the study. No test article was detected in the vehicle control samples. Overall results indicate accuracy of preparation and stability of the formulations used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

 

OECD 471

The test item was tested for mutagenicity in the Ames test and in the E. coli- reverse mutation assay both in the standard plate test and in the preincubation test with and without the addition of a metabolizing system obtained from rat liver (S-9 mix) using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA at a dose of 20-5000 µg/plate (BASF, 1998). Positive control substances caused increases in the number of revertants as expected, indicating proper test conditions. The test substance showed no mutagenic effects at any dose level including the recommended limit dose of 5000 µg/plate.

OECD 473

The test item was assessed for its potential to induce structural chromosome aberrations in primary lymphocytes in vitro in two independent experiments at doses of 125 - 1580 µg/mL with and without metabolic activation (BioReliance, 2007). Under the experimental conditions reported no increased aberration frequencies were observed by the chromosome aberration test. Based on the findings of this in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes, the test item was negative for the induction of structural or numerical chromosome aberrations in both the non-activated and the S9-activated test systems.

OECD 476

The substance was tested with and without metabolic activation for mammalian gene mutation (HPRT locus) in Chinese hamster ovary cells (BASF, 2020). The concentration range tested was up to 1600 µg/mL (without and with metabolic activation by S9-Mix). No induction of forward mutations was observed in any of the experiments. Positive and negative controls gave the expected results. Therefore, the substance is non-mutagenic in CHO cells (HGPRT-/+).

 

In vivo

 

OECD 474

In a GLP guideline study according to OECD 474 (BioReliance, 2007), the test item was tested for clastogenicity and for the ability to induce spindle poison effects in ICR mice using the micronucleus test method. The test substance, dissolved in corn oil was administered by gavage to male and female animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg bw body weight in a volume of 20 mL/kg body weight. As a negative control, corn oil (the selected vehicle) was administered to male mice by the same route, and gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. The test item reduced the ratio of polychromatic erythrocytes to total erythrocytes relative to the respective vehicle controls in both sexes. Although these reductions did not occur in a dose-related manner, they suggest that the test item was bioavailable to the bone marrow target tissue. The test item did not produce a significant increase in the incidence of micronucleated polychromatic erythrocytes in either sex of any group relative to the respective vehicle control groups at either 24 or 48 hours after dose administration. Based on the observations and under the conditions described, the substance was not clastogenic in the mouse micronucleus assay.

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 (EC) No 1272/2008. Based on available data the test item is not classified and labelled for genotoxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.