4,8-Dimethyl-2,5,7,10-tetraoxaundecane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

08 December 2020 to 25 January 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie
- Purity, including information on contaminants, isomers, etc.: 99.90%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, keep under inert atmosphere (nitrogen), in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of 4,8-Dimethyl-2,5,7,10-tetraoxaundecane in the vehicle were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
L5178Y mouse lymphoma (3.7.2c) cells (Clive and Spector, 1975), were obtained from American Type Culture Collection (ATCC), Virginia.

For cell lines:
- Absence of Mycoplasma contamination: Cell stocks are periodically checked for freedom from mycoplasma contamination.
- Periodically ‘cleansed’ of spontaneous mutants: yes; Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 to -150°C, in heatinactivated donor horse serum (HiDHS) containing 10% DMSO

MEDIA USED
R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 g/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R10p medium was used for cell culture unless otherwise specified.
Selective medium consisted of R10p containing 4 g/mL trifluorothymidine (TFT).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was from Molecular Toxicology Inc and stored at -90 to -70°C.
- method of preparation of S9 mix: S9 mix contained: S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0. The co-factors were prepared, neutralised with 1N NaOH and filter sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: the final concentration of the S9 fraction was 2% v/v, if present

Test concentrations with justification for top dose:

The solubility of the test item in a vehicle compatible with this test system was assessed. 4,8-Dimethyl-2,5,7,10-tetraoxaundecane was found to be soluble at 192.25 mg/mL in water. This gave a final concentration of 1922.5 µg/mL (10 mM) when dosed at 1% v/v.
Preliminary toxicity test: 3.75, 7.51, 15.02, 30.04, 60.08, 120.16, 240.31, 480.63, 961.25 and 1922.5 µg/mL
Mutation tests: 3 hours +/- S9 mix: 120.16, 240.31, 480.63, 961.25 and 1922.5 µg/mL

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate cultures were prepared throughout for all cultures.
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cultures contained a total of 12 x 106 cells in a final volume of 10 mL
- Test substance added in medium

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Following the 3-hour treatment, the cells were washed once, re-suspended in R10p to nominally 2 x 10^5 cells/mL assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype.
- Selection time (if incubation with a selective agent): Mutant potential was assessed by plating 2 x 10^3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 34 to 39°C in an atmosphere of 5% CO2 in air. After the plates had been incubated for 10 to 12 days, the number of empty wells was assessed for each 96-well plate (P0).
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: Trifluorothymidine is used at 4 µg/ml in the selective medium for 48 hours
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 12 x 10^6 cells in a final volume of 10ml - After 48 hours cultures with a density of more than 1 x 10^5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions: GEF = Global Evaluation Factor. For microwell assays this is 126 x 10^-6. Providing that all acceptability criteria were fulfilled, the test item was considered to be clearly positive if, in any of the experimental conditions examined the increase in MF above the concurrent background exceeded the GEF and the increase was concentration related (i.e., there is a significant positive linear trend). The test item is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, the test item was considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test item is then considered unable to induce mutations in this test system. If the maximum concentration was based on cytotoxicity, the highest concentration aimed to achieve between 20 and 10% RTG. The consensus is that care should be taken when interpreting positive results only found between 20 and 10% RTG and a result would not be considered positive if the increase in MF occurred only at or below 10% RTG (if evaluated).
There are some circumstances under which additional information may assist in determining that a test item is not mutagenic when there is no culture showing an RTG value between 20 and 10% RTG. These situations are outlined as follows:
(1) There was no evidence of mutagenicity (e.g., no dose response, no mutant frequencies above those seen in the concurrent negative control or historical background ranges, etc.) in a series of data points within 100% to 20% RTG and there was at least one data point between 20 and 25% RTG.
(2) There was no evidence of mutagenicity (e.g., no dose response, no mutant frequencies above those seen in the concurrent negative control or historical background ranges, etc.) in a series of data points between 100% to 25% RTG and there was also a negative data point slightly below 10% RTG. In both of these situations the test item is concluded to be negative.

Statistics:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users), which follows the methods described by Robinson et al. (1989). Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

No precipitate was observed by eye at the end of treatment. Exposure at concentrations from 3.75 to 1922.5 µg/mL in the absence and presence of S9 mix (3-hour exposure) resulted in RSG values from 120 to 54% and from 128 to 72% respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

It was concluded that 4,8-Dimethyl-2,5,7,10-tetraoxaundecane did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

4,8-Dimethyl-2,5,7,10-tetraoxaundecane was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase
deficient genotype (TK-/-).
The study consisted of a preliminary toxicity test and two independent mutagenicity assays. The cells were exposed for 3 hours in the absence or presence of exogenous metabolic activation (S9 mix).
4,8-Dimethyl-2,5,7,10-tetraoxaundecane was found to be soluble at 192.25 mg/mL in water.
A final concentration of 1922.5 g/mL (10 mM), dosed at 1%v/v was used as the maximum concentration in the preliminary toxicity test. This is the standard limit concentration within this test system as recommended in the current OECD Guideline 490 (2016).
In the preliminary toxicity test following a 3-hour exposure to 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at concentrations from 3.75 to 1922.5 µg/mL, relative suspension growth (RSG) was reduced from 120 to 54% and from 128 to 72% in the absence and presence of S9 mix respectively. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to test up the standard limit concentration within this test system as recommended in the regulatory guidelines.
In the main test following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in both the 3-hour treatment in the absence and presence of S9 mix was 1922.5 g/mL. No significant reduction in relative total growth (RTG) was observed in either the absence and presence of S9 mix.
In all tests the concurrent vehicle and positive control were within acceptable ranges.
It was concluded that 4,8-Dimethyl-2,5,7,10-tetraoxaundecane did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 2020 to 13 April 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie
- Purity, including information on contaminants, isomers, etc.: 99.90%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, keep under inert atmosphere (nitrogen)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The solubility of the test item in a vehicle compatible with this test system was assessed. 4,8-Dimethyl-2,5,7,10-tetraoxaundecane was found to be soluble at 199.25 mg/mL in water. This gave a final concentration of 1922.5 μg/mL (10 mM) when dosed at 1% v/v.

Species / strain / cell type:

lymphocytes: cultured human peripheral blood lymphocytes in vitro

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
- Suitability of cells:
- Normal cell cycle time (negative control):

For lymphocytes:
- Sex, age and number of blood donors: Human blood was collected aseptically from two healthy, non-smoking, adult (between 22-35 years of age) donors with no known illness or recent exposures to genotoxic agents, pooled (in equal volumes from each donor). Blood was pooled from two females (aged 28 and 25) for the preliminary toxicity test, two females (aged 23 and 26) for the main micronucleus test, two females (aged 26 and 22) for the additional main test, and from one male and one female (aged 35 and 23) for the second additional main test.
- Whole blood cultures were established by placing 0.4 mL of pooled blood and 0.2 mL PHA into 8.3 mL of HML media, so that the final volume following addition of S9 mix/KCl and the test item in the chosen vehicle was 10 mL. All cultures were then incubated at 34 to 39°C, and the cells were re-suspended (twice daily) by gentle inversion. Historically the average donor doubling time for this laboratory was ca. 13 hours and the study design is based upon this.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: HML Media RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine.

Cytokinesis block (if used):

Cytochalasin B at final concentration of 6 µg/ml; duration of cell exposure: 3 and 20 hours

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver
- method of preparation of S9 mix: The S9 mix contained: S9 fraction (10% v/v), NaH2PO4 . H2O (15.5 mM), Na2HPO4 (84.8 mM), MgCl2 . 6H2O (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), NADP disodium salt (4 mM).
- concentration or volume of S9 mix and S9 in the final culture medium: 1mL of 10% S9 mix was added to the appropriate cultures so that S9 homogenate was present at a final concentration of 1% v/v.

Test concentrations with justification for top dose:

Preliminary toxicity test: 3.75, 7.51, 15.02, 30.04, 60.08, 120.16, 240.31, 480.63, 961.25 and 1922.5 μg/mL
Main tests: 3 hours (+/-S9 mix): 240.31, 480.63, 961.25 and 1922.5 μg/mL; 20 hours (-S9 mix): 240.31, 480.63, 961.25 and 1922.5 μg/mL
Additional main tests: 20 hours (-S9 mix): 480.63, 720.94, 961.25, 1494.38 and 1922.5 μg/mL
Justification for top dose: The solubility of the test item in a vehicle compatible with this test system was assessed. 4,8-Dimethyl-2,5,7,10-tetraoxaundecane was found to be soluble at 199.25 mg/mL in water. This gave a final concentration of 1922.5 μg/mL (10 mM) when dosed at 1% v/v.

Vehicle / solvent:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Single cultures were prepared for each treatment level and duplicate cultures were prepared for vehicle controls.
- Number of independent experiments: 3


TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 or 20 hours
- Harvest time after the end of treatment (sampling/recovery times): The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 34 to 39°C, cultures were then incubated for 3 minutes at 34 to 39°C to cause swelling. No recovery

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: CytoB at final concentration of 6 µg/ml; duration of cell exposure: 3 and 20 hours
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cultures were centrifuged at 500 g for 5 minutes, the supernatant removed, and the cell pellet re-suspended in the residual supernatant. Pre-cleaned microscope slides were prepared for each culture by aliquoting 50 μL of the cell suspension onto the slides and allowing the slides to air-dry. One slide was prepared from each culture. The slides were then stained using an acridine orange solution at 0.0125 mg/mL in purified water. The remaining cell cultures were stored at 2 to 8°C until slide analysis was complete.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Determination of polyploidy: mononucleate, binucleate and polynucleate cells assessed

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytokinesis block proliferative index

Evaluation criteria:

Providing that all of the acceptance criteria have been met, the test item was considered to be clearly positive if, in any of the experimental conditions examined:
At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
Any of the results are outside the distribution of the historical vehicle control data (above the upper 95% confidence limit).
If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.
Providing that all of the acceptance criteria have been met, a negative response will be claimed if, in all of the experimental conditions examined:
None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
There is no concentration-related increase when evaluated with an appropriate trend test.
All results are inside the distribution of the historical vehicle control data (below the 95% confidence limit).
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.

Statistics:

The analysis assumed that the replicate was the experimental unit. An arcsine square-root transformation was used to transform the data. 4,8-Dimethyl-2,5,7,10-tetraoxaundecane treated groups were then compared to control using Williams’ tests (Williams 1971, 1972). Positive controls were compared to control using two-tailed t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups.
Statistical significance was declared at the 5% level for all tests.
Data were analyzed using SAS (SAS Institute 2002) and Quasar (Quasar 1.5 2017).

Key result

Species / strain:

lymphocytes: cultured human peripheral blood lymphocytes in vitro

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A reduction in the CBPI compared to vehicle control values equivalent to 9.3% cytostasis, was obtained with 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at 1922.5 μg/mL (10 mM).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: cultured human peripheral blood lymphocytes in vitro

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A reduction in the CBPI compared to vehicle control values equivalent to 27.3% cytostasis, was obtained with 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at 1922.5 μg/mL (10 mM).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: 20-hour exposure - additional treatment

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A reduction in the CBPI compared to vehicle control values equivalent to 10.6% cytostasis, was obtained with 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at 1922.5 μg/mL (10 mM).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not valid

Key result

Species / strain:

lymphocytes: cultured human peripheral blood lymphocytes in vitro

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3-h treatment : A reduction in the CBPI compared to vehicle control values equivalent to 4.9% (absence of S9 mix) or 12.0% (presence of S9 mix) cytostasis, was obtained with 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at 1922.5 μg/mL (10 mM).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

It was concluded that 4,8-Dimethyl-2,5,7,10-tetraoxaundecane did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Executive summary:

1.1 Study Design
This study was designed to assess the potential of 4,8-Dimethyl-2,5,7,10-tetraoxaundecane to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro.
The study consisted of a preliminary toxicity test and a main micronucleus test and two additional tests for 20 hours in the absence of S9 mix. Human lymphocytes in whole blood culture, were exposed to the test item for 3 hours in both the absence and presence of exogenous metabolic activation (S9 mix) and for 20 hours in the absence of S9 mix. The maximum final concentration to which the cells were exposed was 1922.5 μg/mL, dosed at 1% v/v, in order to test up to 10 mM, the maximum concentration as recommended in the current OECD Guideline 487 (2016). Vehicle (water) and positive control cultures were included in all appropriate test conditions.
1.2 Results
Three 4,8-Dimethyl-2,5,7,10-tetraoxaundecane concentrations were assessed for determination of induction of micronuclei. The highest concentration selected (1922.5 μg/mL; 10 mM) was the maximum concentration as recommended in the current OECD Guideline 487 (2016) as no significant cytostasis was observed and no precipitate was observed at the end of treatment.
Following 3-hour treatment in the absence of S9 mix, a reduction in the CBPI equivalent to 4.9% cytostasis was obtained with 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at 1922.5 μg/mL (10 mM). Concentrations of 4,8-Dimethyl-2,5,7,10-tetraoxaundecane selected for micronucleus analysis were 480.63, 961.25 and 1922.5 μg/mL. 4,8-Dimethyl-2,5,7,10-tetraoxaundecane caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear concentration relationship. The mean micronucleus frequencies for the vehicle and test item treated cultures were within the laboratory historical 95% confidence limits. Therefore, this experiment was concluded to be clearly negative.
Following 3-hour treatment in the presence of S9 mix, a reduction in the CBPI equivalent to 12.0% cytostasis was obtained with 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at 1922.5 μg/mL (10 mM). Concentrations of 4,8-Dimethyl-2,5,7,10-tetraoxaundecane selected for micronucleus analysis were 480.63, 961.25 and 1922.5 μg/mL. 4,8-Dimethyl-2,5,7,10-tetraoxaundecane caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear concentration relationship. The mean micronucleus frequencies for the vehicle and test item treated cultures were within the laboratory historical control data (HCD) 95% confidence limits. Therefore, this experiment was concluded to be clearly negative.
Following a 20-hour exposure in the absence of S9 mix, a reduction in the CBPI equivalent to 9.3% cytostasis was obtained with 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at 1922.5 μg/mL (10 mM). Concentrations of 4,8-Dimethyl-2,5,7,10-tetraoxaundecane selected for micronucleus analysis were 480.63, 961.25 and 1922.5 μg/mL. 4,8-Dimethyl-2,5,7,10-tetraoxaundecane caused no statistically significant increases in the number of binucleate cells containing micronuclei. There was evidence of a linear dose-concentration relationship at 961.25 μg/mL (p=0.015). Following an additional analysis of 1000 cells from the vehicle and test item treated concentrations there was evidence of a linear concentration relationship at 961.25 μg/mL (p=0.028). The mean micronucleus frequencies for the vehicle and test item treated cultures were within the laboratory HCD 95% confidence limits. The criteria for a negative response was not met and therefore the treatment was considered to be equivocal and an additional test was performed to investigate the reproducibility of the trend.
Following a second additional 20-hour exposure in the absence of S9 mix, a reduction in the CBPI equivalent to 27.3% cytostasis was obtained with 4,8-Dimethyl-2,5,7,10-tetraoxaundecane at 1922.5 μg/mL (10 mM). Concentrations of 4,8-Dimethyl-2,5,7,10-tetraoxaundecane selected for micronucleus analysis were 480.63, 961.25 and 1922.5 μg/mL. 4,8-Dimethyl-2,5,7,10-tetraoxaundecane caused no statistically significant increases in the number of binucleate cells containing micronuclei and there was no evidence of a linear concentration relationship. The mean micronucleus frequencies for the vehicle and test item treated cultures were within the laboratory HCD 95% confidence limits. Therefore, this experiment and exposure condition was concluded to be clearly negative.
The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.
1.3 Conclusion
It was concluded that 4,8-Dimethyl-2,5,7,10-tetraoxaundecane did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 August 2016 to 25 August 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Lambiotte & Cie - 1604211010R
- Purity, including information on contaminants, isomers, etc.: 99.97%

Target gene:

histidine or tryptophan locus

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:

Type and composition of metabolic activation system:
- source of S9: The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 05 June 2016 was used in this study.
- method of preparation of S9 mix: The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test. A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 ml; 10% S9 fraction in S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 certificate of efficacy provided by the supplier

Test concentrations with justification for top dose:

The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.
The highest concentration of the test item tested in this study was 50 mg/mL (final plate concentration was 5000 μg/plate). The test item was accurately weighed and diluted in sterile distilled water by mixing on a vortex mixer on the day of each experiment. The highest concentration in each test was diluted with sterile distilled water to produce a series of lower concentrations, separated by approximately half-log10 intervals. No correction was made for purity.
Concentrations tested for plate incorporation method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg
Concentrations tested for pre-incubation method: 15, 50, 150, 500, 1500 and 5000 µg

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water.

- Justification for choice of solvent/vehicle: The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration triplicate
- Number of independent experiments 2 (plate incorporation and pre-incubation)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) and in media (pre-incubation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 hours
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The plates were viewed microscopically for evidence of thinning

Rationale for test conditions:

Experiment 1 (plate incorporation method): The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Experiment 2 (pre-incubation method): The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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

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:

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

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

4,8-Dimethyl-2,5,7,10-tetraoxaundecane was considered to be non-mutagenic in the tested species under the conditions of this test.

Executive summary:

Introduction
The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Results
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Conclusion
4,8-Dimethyl-2,5,7,10-tetraoxaundecane was considered to be non-mutagenic in the tested species under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)