Dihexyl ether

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 bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 27, 2018 to September 24, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction, derived from male Wistar rats, phenobarbital (80 mg/kg bw) / ß-naphthoflavone (100 mg/kg bw) induced for three consecutive days by oral route

Test concentrations with justification for top dose:

Pre-experiment: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 µL/plate

Experiment I:
TA98 with/without metabolic activation and TA100 with metabolic activation: 3.16, 10.0, 31.5, 100, 316, 1000, 2500, 5000 nl/plate
TA100, TA1535, TA 1537 and TA 201 without metabolic activation: 0.036, 0.0100, 0.316, 1.00, 3.16, 10, 31.6, 100, 316 nL/plate
TA1535, TA1537, TA 102 with metabolic activation: 1.00, 3.16, 10, 31.6, 100, 316, 1000 and 2500 nL/plate

Experiment II:

without metabolic activation: 0.0632, 0.200, 0.632, 2.00, 6.32, 20.0, 63.2 and 200 nL/plate
with metabolic activation: 0.632, 2.00, 6.32, 20.0, 63.2, 200, 632 and 2000 nL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-Aminoanthacene: 2.5 µg/plate in DMSO for TA98, TA100, TA1535, TA 1537, TA102 with met. activ.; 10 µg/plate in DMSO for TA102 with met. activ. 4-nitro-o-phenylene-diamine: 10 µg/plate for TA98 without met. activ.; 40 µg/plate for TA1537 without met.act.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approx. ≤ 0.5 in relation to the solvent control

OTHER:
The properties of the S. typhimurium with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al..
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH, Germany). Tester strains with a low spontaneous mutation frequency (TA 1535 and TA 1537) were counted manually.

Evaluation criteria:

Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean
values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice
as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
According to the OECD guidelines, the biological relevance of the results is the criterion for the
interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible
biologically relevant positive response at any of the dose groups is considered to be non-mutagenic
in this system.
Criteria of validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, TA102)
- the negative control plates (A. dest.) with and without S9 mix are within the historical control data ranges
-corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.
- at least five different concentrations of each tester strain are analysable.

Statistics:

A statistical evaluation of the results was not regarded as necessary as the biological relevance of the results is the criterion for the interpretation of results (according to guideline).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic effects observed in exp. I at 31.6 nl/plate and higher without metab. activ. and at 1000 nl/plate and higher with metab. activ, In exp. II at 63.2 nl/plate and higher without metab. activ. and at 2000 nl/plate with metab. activ.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Remarks:

Toxic effects observed in exp. I at 31.6 nl/plate and higher without metab. activ. and at 1000 nl/plate and higher with metab. activ, In exp. II at 63.2 nl/plate and higher without metab. activ. and at 316 nl/plate and higher with metab. activ.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Remarks:

Toxic effects observed in exp. I at 31.6 nl/plate and higher without metab. activ. and at 1000 nl/plate and higher with metab. activ, In exp. II at 63.2 nl/plate and higher without metab. activ. and at 2000 nl/plate with metab. activ.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Remarks:

Toxic effects observed in exp. I at 10.0 nl/plate and higher without metab. activ. and at 100 nl/plate and higher with metab. activ, In exp. II at 6.32 nl/plate and higher without metab. activ. and at 2000 nl/plate with metab. activ.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic effects observed in exp. I at 100 nl/plate and higher without metab. activ. and at 2500 nl/plate with metab. activ, In exp. II at 63.2 nl/plate and higher without metab. activ. and at 2000 nl/plate with metab. activ.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No recipitation of the test item was observed in any tester strains used in experiment I and II (with and without metabolic activation).

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determind with tester stains TA 98 and TA 100. Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The control plates with and without S9 mix were within the historical control data range.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance was considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item dihexyl ether was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I and experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. Precipitation of the test item was not observed in any tester strains used in experiment I and II (with and without metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with dihexyl ether at any concentration level tested, neither in the presence nor absence of metabolic activation in experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the two independent experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, dihexyl ether did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, dihexyl ether is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 October - 14 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 03 Aug 2018

Type of assay:

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

Target gene:

Thymidine kinase (tk) locus in L5178Y 3.7.2 C mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y TK+/- 3.7.2 C mouse lymphoma ; American Type Culture Collection

For cell lines:
- Absence of Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically ‘cleansed’ of spontaneous mutants: no

MEDIA USED
- Three types of RPMI 1640 medium
RPMI-5: 5% v/v horse serum, antibiotic-antimycotic solution, pluronic-F68 and pyruvic acid
RPMI-10: 10% v/v horse serum, antibiotic-antimycotic solution, pluronic-F68 and pyruvic acid
RPMI-20: 20% v/v horse serum, antibiotic-antimycotic solution and pyruvic acid

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day (for both inducers). For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%.

Test concentrations with justification for top dose:

Pre-liminary toxicity test (with and without S9-mix, 3 hour treatment, without S9-mix, 24 hour treatment): highest concentration was 2000 μg/mL

Main test 1:
3 hour treatment (with S9-mix): 15.625, 31.25, 62.5, 125, 250, 300, 350, 400 μg/mL
3 hour treatment (without S9-mix): 5, 10, 20, 40, 45, 50 μg/mL

Main test 2:
3 hour treatment (with S9-mix): 15.625, 31.25, 62.5, 125, 250, 300, 350, 400 μg/mL
24 hour treatment (without S9-mix): 2.5, 5, 10, 15, 20, 25, 30 μg/mL

Vehicle / solvent:

- Vehicle for the test substance: Acetone
- Vehicle for the positive control: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

4 μg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.15 μg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): For the 3-hour treatments, 10^7 cells were placed in each of a series of 75 cm2 sterile flasks. For the 24-hour treatment, 6x10^6 cells were placed in each of a series of 25 cm2 sterile flasks.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 or 24 hours
- Harvest time after the end of treatment (sampling/recovery times): To allow expression of TK- mutations, cultures were maintained in flasks for 2 days. On each day, cell density was adjusted to a concentration of 2x10^5 cells/mL (whenever possible) and transferred to flasks for further growth.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Method used: Plating for -trifluorothymidine (TFT) resistance
- Selective agent: At the end of the expression period, the cell concentration was adjusted to 1x10^4 cells/mL. TFT (300 μg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: Averaging 1.6 cells per well (96-well plate) for viability and 2x10^3 cells per well for TFT resistance.
- Criteria for small (slow growing) and large (fast growing) colonies: Not indicated

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative Total Growth (RTG)
RSG = [SG1(test) x SG2(test)] / [SG1(control) x SG2(control)]
RTG = RSG x RCE

Acceptability criteria
The assay was considered valid if all of the following criteria were met (based on the relevant guidelines):
1. The mutant frequency in the negative (vehicle/solvent) control cultures fell within the normal range (50-170 mutants per 10^6 viable cells).
2. The positive controls met at least one of the following two criteria:
-The positive control chemical demonstrated an absolute increase in total MF that is, an increase above the spontaneous background MF of at least 300 x 10^-6. At least 40% of the IMF reflected in the small colony MF.
-The positive control substance had an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent untreated/vehicle control (a small colony IMF of 150 x 10^-6).
3. The plating efficiency (PEviability) of the negative (vehicle) controls was within the range of 65% to 120% at the end of the expression period.
4. At least four test concentrations were present, where the highest concentration produced approximately 80-90% toxicity (measured by RTG), resulted in precipitation, or it was 2 mg/mL, 2 μL/mL or 0.01 M (whichever is the lowest), or it was the highest practical (achievable) concentration.

Evaluation criteria:

Evaluation criteria
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.
The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. Caution was exercised with positive results obtained at levels of cytotoxicity lower than 10% (as measured by RTG).

Statistics:

The negative (vehicle/solvent) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no large changes after treatment
- Data on osmolality: no large changes after treatment
- Water solubility: insolubility was observed in the final treatment medium at the end of the treatment with metabolic activation at concentration range of 250-400 μg/mL. No insolubility was observed in the final treatment medium at the end of the treatment without metabolic activation.
- Precipitation and time of the determination: Visual examination for precipitation of test item in the final culture medium was conducted at the beginning and end of the treatments. Highest conecntration resulted in precipitation.

VALIDITY OF MUTATION ASSAYS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently in the study. The spontaneous mutation frequency of the negative (vehicle/solvent) and untreated controls were in the recommended range (50-170 x 10^-6) in all cases.
The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays. All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD No. 490 criteria.
The plating efficiencies for the negative (vehicle/solvent) controls of the test item and positive control item as well as the untreated control samples at the end of the expression period (PEviability) were acceptable in all assays.
The number of test concentrations evaluated was at least four in Assay 1 and Assay 2, which met the acceptance criteria about the minimum number of evaluated concentrations.
The tested concentration range in the study was considered to be adequate as the highest evaluated concentration showed proper degree of cytotoxicity (approximately 80-90%, i.e. approximately 10-20 relative total growth)*. Lower test concentrations were usually spaced by a factor of two, but more closely spaced concentration were used in the expected cytotoxic range in all cases in an attempt to obtain values in the range of 10-20% viability.
*Note:
In Assay 1 with metabolic activation the relative total growth of the highest evaluated concentration (350 μg/mL, replicate “A”) was 28%, which was slightly above the target range according to the OECD guideline, however in Assay 2 at this concentration the relative total growth value was within the target range (RTG was 12%).Thus, the results were considered to cover appropriate concentrations, being acceptable to justify the study for the exposures with metabolic activation.
In Assay 1 without metabolic activation the relative total growth of the highest evaluated concentration (40 μg/mL) was 59%, and in Assay 2 without metabolic activation the relative total growth of the highest evaluated concentration (25 μg/mL) was <1% and the next evaluated concentration (20 μg/mL) was 52%(a very steep dose response). A lower or higher degree of cytotoxicity was observed in each case than the recommendation of the relevant OECD guideline.
However, in Assay 1 no cells survived at 50 and 45 μg/mL and in Assay 2 no cells survived at 30 μg/mL or excessive cytotoxicity was observed at 25 μg/mL concentrations. A closely spaced concentration range was used to properly cover concentrations from cytotoxicity to no cytotoxicity, and the results showed a negative response in both short and long treatment without metabolic activation. Therefore, taking together the two experiments without metabolic activation, it is concluded that it is valid to state that the test item is negative under this condition.
Suspension growth value of the untreated and negative (vehicle/solvent) control samples were in line with the recommended range in all cases (i.e. 8-32 fold in case of short treatments and 32-180 fold in case of long treatment).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Cyclophosphamide 3hS9+ [Min. 196.1; Max. 2642.5]
4-Nitroquinoline-N-oxide 3hS9- [Min. 223.5; Max. 1687.3], 24hS9- [Min. 245.0; Max. 1577.6]
- Negative (solvent/vehicle) historical control data:
Culture medium 3hS9+ [Min. 39.2; Max. 198.5], 3hS9- [Min. 52.6; Max. 235.6], 24hS9- [Min. 41.7; Max. 179.1]
Distilled water 3hS9+ [Min. 33.4; Max. 121.8], 3hS9- [Min. 55.1; Max. 125.0], 24hS9- [Min. 43.2; Max. 141.1]
DMSO 3hS9+ [Min. 44.2; Max. 269.9] 3hS9- [Min. 33.7; Max. 261.6], 24hS9- [Min. 47.1; Max. 159.4]

ASSAY 1

A 3-hour treatment with metabolic activation and a 3-hour treatment without metabolic activation were performed. Treatment concentrations were 400, 350, 300, 250, 125, 62.5, 31.25 and 15.625 μg/mL with metabolic activation and 50, 45, 40, 20, 10 and 5 μg/mL without metabolic activation.

In the presence of S9-mix, cytotoxicity was seen at concentration range of 62.5-400 μg/mL. No cells survived the expression period in the samples of 400 μg/mL concentration. An evaluation was made using data of seven concentrations.

In the absence of S9-mix (3-hour treatment), cytotoxicity of the test item was observed at higher concentrations (concentration range of 40-50 μg/mL), no cells survived the expression period in the samples of 50 and 45 μg/mL concentrations, however lower degree of cytotoxicity was observed at 40 μg/mL (RTG value of the highest evaluated concentration was 59%) and no cytotoxicity was observed at further concentrations. An evaluation was made using data of four concentrations.

ASSAY 2

A 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 400, 350, 300, 250, 125, 62.5, 31.25 and 15.625 μg/mL with metabolic activation, and 30, 25, 20, 15, 10, 5 and 2.5 μg/mL concentrations without metabolic activation.

In the presence of S9-mix (3-hour treatment), cytotoxicity was seen at concentration range of 62.5-400 μg/mL. Excessive cytotoxicity was observed at 400 μg/mL. An evaluation was made using data of seven concentrations (concentration range of 15.625-350 μg/mL). Relative total growth of the highest evaluated concentration (350 μg/mL) was within the target range (RTG value of 12%). There was statistically significant increase in the mutation frequency values in the highest evaluated concentration, but it was statistically borderline. No statistically significant increase was observed at further concentrations.

In the absence of S9-mix (24-hour treatment), cytotoxicity of the test item was observed at concentration range of 20-30 μg/mL. No cells survived the expression period in the samples of 30 μg/mL concentration and excessive cytotoxicity was observed at 25 μg/mL concentration. An evaluation was made using data of six concentrations (concentration range of 2.5-25 μg/mL). Relative total growth of the highest evaluated concentration (25 μg/mL was <1%) and the RTG value of the next evaluated concentration (20 μg/mL) was 52%, thus the range was covered between <1% and 52%.

CONTROLS

Untreated, negative (vehicle/solvent) and positive controls were run concurrently in the study. The spontaneous mutation frequency of the negative (vehicle/solvent) and untreated controls were in the recommended range (50-170 x 10-6) in all cases.

The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays. All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD No. 490 criteria.

The plating efficiencies for the negative (vehicle/solvent) controls of the test item and positive control item as well as the untreated control samples at the end of the expression period (PEviability) were acceptable in all assays.

Conclusions:

An in vitro mammalian cell gene mutation test was performed according to OECD guideline 490 and in accordance with GLP principles. No mutagenic effect of Di-n-hexyl ether was observed in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

An in vitro study was conducted to assess the gene mutation in the mouse lymphoma test (Varga-Kanizsai B; 2019) in two assays.

In Assay 1, following a 3-hour treatment with metabolic activation, cytotoxicity was seen at several concentrations up to 400 μg/mL. In Assay 1 with metabolic activation no cells survived the expression period in the samples of 400 μg/mL concentration and in replicate “B” sample of the 350 μg/mL concentration. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.

In Assay 1, following a 3-hour treatment without metabolic activation, cytotoxicity of the test item was observed at higher concentrations (concentration range of 40-50 μg/mL). No cells survived the expression period in the samples of 50 and 45 μg/mL concentrations. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.

In Assay 2, following a 3-hour treatment with metabolic activation, cytotoxicity was seen at several concentrations up to 400 μg/mL. Relative total growth of the highest evaluated concentration (350 μg/mL) was within the target range (RTG value of 12%). There was statistically significant increase in the mutation frequency values of the highest evaluated concentration. A dose response was indicated by the linear trend analysis, but based on the individual values biologically it was not relevant (the difference did not exceed the Global Evaluation Factor, GEF).

This experiment was considered as being negative and confirmed the negative result of the first test with metabolic activation.

In Assay 2, following a 24-hour treatment without metabolic activation, cytotoxicity of the test item was observed at higher concentrations concentration range of (20-30 μg/mL). No cells survived the expression period in the samples of 30 μg/mL concentration and excessive cytotoxicity was observed at 25 μg/mL concentration. Relative total growth of the highest evaluated concentration (25 μg/mL was <1%) and the RTG value of the next evaluated concentration (20 μg/mL) was 52%, thus the range was covered between <1% and 52%. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

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:

August 2018 to December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was performed for the purpose of notification in another region requesting this study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

temp. value (18.1°C - 25.2°C) and rel. humidity value (max. of 77%) outside the expected ranges were recorded occasionally times during the study. These differences were considered not to adversely affect the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH (Address: Sandhofer Weg 7, Sulzfeld D-97633, Germany)
- Age at study initiation: Approximately 7 or 8 weeks (preliminary experiments) or 9 weeks (main test) at the treatment
- Weight at study initiation: 36.5 – 40.3 g (males, preliminary experiment I.)
29.0 – 32.0 g (females, preliminary experiment I.)
36.2 – 38.5 g (males, preliminary experiment II.)
30.0 – 31.5 g (females, preliminary experiment II.)
30.5 – 32.6 g (females, preliminary experiment III.)
26.1 – 29.4 g (females, main test)
The weight variation did not exceed ± 20 percent of the mean weight/sex at the start of the treatment.
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Type II polypropylene/polycarbonate cages
- Diet (e.g. ad libitum): ssniff(R) SM R/M "Autoclavable complete diet for rats and mice ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1-25.2°C (target: 22 ± 3°C)
- Humidity (%): 32-77% (target: 30-70%)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 22, 2019 To: September 12, 2019

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil
- Amount of vehicle (if gavage or dermal): 5 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The necessary amount of the test item was weighed into a calibrated volumetric flask or other appropriate glass container; the required volume of vehicle was added and mixed using a magnetic stirrer to obtain homogenous formulations. The concentrations of the test item formulations were selected to assure the same dosing volumes in mice for all dose levels (the treatment volume was 5 mL/kg bw in case of the negative control (in the preliminary toxicity test II. and III.) and the test item treated groups in the preliminary experiments; the treatment volume was 5 mL/kg bw in case of the negative control and the test item treated groups and 10 mL/kg bw in case of the positive control group in the main study). All the dose levels were expressed in terms of the weight of the test item in the formulations.

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5
High dose group: 5+2 (Two additional mice were dosed in the High dose group to replace any animal which may die before the scheduled sacrifice time or serve as additional sample in case of borderline results. One additional animal was used for evaluation, because one animal was found dead in the High dose group before necropsy (Day 15). Bone marrow smears were also prepared from the other additional mouse, but those slides were not evaluated.)

Control animals:

yes

Positive control(s):

Cyclophosphamide at 60 mg/kg bw (10 ml/kg bw) at one occasion by the ip route

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Bone marrow was obtained from two exposed femurs of each surviving mouse immediately after sacrifice. The bone marrow was flushed into a sterile centrifuge tube from of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (3 slides / animal). (Note:Bone marrow smears were prepared from the replacement mice. The bone marrow smears of one of the additional animal of High dose group was scored as it was necessary for the proper evaluation of the study. The bone marrow smears of the other additional animal were not scored. However, all the processed slides will be archived in the raw data.) Slides were air-dried at room temperature for approximately 24 hours. Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Slides were stained with 10 % Giemsa solution for 18 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature overnight. After staining, coverslips were mounted on them.

Evaluation criteria:

Criteria for Identification of Micronucleated Erythrocytes:

- A bluish mauve strongly coloured uniform round or oval particle in the cell.
- The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
- During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
- The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.

The Micronucleus Test was considered valid if it met the following criteria:

- the frequency of micronucleated polychromatic erythrocytes found in the negative (vehicle) control was within the range of historical laboratory control data,
- the positive control item produced biologically relevant increase in the number of micronucleated polychromatic erythrocytes,
- each treated and control group included at least 5 analysable animals.

Statistics:

Kruskal-Wallis Non Parametric ANOVA test at the Test Site

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Minor clinical signs related to treatment were observed considered to be treatment related. Increased in the liver weights by 44.2% in the High dose were observed, clearly demonstrating a systemic dose related effect as proof of systemic exposure.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: up to 2000 mg/kg bw/d
- Solubility: soluble in vehile up to 400 mg/ml
- Clinical signs of toxicity in test animals:
- Evidence of cytotoxicity in tissue analysed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay):
- Appropriateness of dose levels and route:
- Statistical evaluation:

Conclusions:

Di-n-hexyl ether test item was shown to have significant systemic exposure, it did not cause statistically or biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in mice. Thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Executive summary:

An in vivo mammalian erythrocyte micronucleus test was conducted according to OECD guideline 474 (Varga-Kanizsai B; 2019). NMRI mice received daily oral doses of 500, 1000 or 2000 mg/kg bw for 14 days. The highest dose level was chosen based on the results of preliminary studies. Minor clinical signs related to treatment were observed which were considered to be treatment related. Increaseds in the liver weights by 44.2% in the High dose were observed, clearly demonstrating a systemic dose related effect as proof of systemic exposure. Treatment with the test item did not affect the ratios of polychromatic erythrocytes among total erythrocytes. No statistically significant or biologically relevant increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) were seen for test item treated mice in any dose groups at any sampling time when compared to the corresponding negative (vehicle) control values. In conclusion, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Two in vitro studies were conducted:

- Bacterial Reverse Mutation Assays (Ames test)

- Gene mutation assay in mouse lymphoma L5178Y TK+/- cells

In the Ames test (Schreib G; 2018), the results were negative for mutagenicity in all tester strains. Cytotoxicity was observed with and without metabolic activation.

A second in vitro study was conducted to assess the gene mutation in the mouse lymphoma test (Varga-Kanizsai B; 2019) in two assays.

In Assay 1, following a 3-hour treatment with metabolic activation, cytotoxicity was seen at several concentrations up to 400 μg/mL. In Assay 1 with metabolic activation no cells survived the expression period in the samples of 400 μg/mL concentration and in replicate “B” sample of the 350 μg/mL concentration. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.

In Assay 1, following a 3-hour treatment without metabolic activation, cytotoxicity of the test item was observed at higher concentrations (concentration range of 40-50 μg/mL). No cells survived the expression period in the samples of 50 and 45 μg/mL concentrations. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.

In Assay 2, following a 3-hour treatment with metabolic activation, cytotoxicity was seen at several concentrations up to 400 μg/mL. Relative total growth of the highest evaluated concentration (350 μg/mL) was within the target range (RTG value of 12%). There was statistically significant increase in the mutation frequency values of the highest evaluated concentration. A dose response was indicated by the linear trend analysis, but based on the individual values biologically it was not relevant (the difference did not exceed the Global Evaluation Factor, GEF).

This experiment was considered as being negative and confirmed the negative result of the first test with metabolic activation.

In Assay 2, following a 24-hour treatment without metabolic activation, cytotoxicity of the test item was observed at higher concentrations concentration range of (20-30 μg/mL). No cells survived the expression period in the samples of 30 μg/mL concentration and excessive cytotoxicity was observed at 25 μg/mL concentration. Relative total growth of the highest evaluated concentration (25 μg/mL was <1%) and the RTG value of the next evaluated concentration (20 μg/mL) was 52%, thus the range was covered between <1% and 52%. No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being negative.

In vivo

An in vivo mammalian erythrocyte micronucleus test was conducted according to OECD guideline 474 (Varga-Kanizsai B; 2019). NMRI mice received daily oral doses of 500, 1000 or 2000 mg/kg bw for 14 days. The highest dose level was chosen based on the results of preliminary studies. Minor clinical signs related to treatment were observed which were considered to be treatment related. Increased in the liver weights by 44.2% in the High dose were observed, clearly demonstrating a systemic dose related effect as proof of systemic exposure. Treatment with the test item did not affect the ratios of polychromatic erythrocytes among total erythrocytes. No statistically significant or biologically relevant increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) were seen for test item treated mice in any dose groups at any sampling time when compared to the corresponding negative (vehicle) control values. In conclusion, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Short description of key information:

In vitro  

Bacterial reverse mutation test (Schreib G. 2018) with dihexyl ether - negative for mutagenicity with and without metabolic activation.  

In vitro gene mutation in mouse lymphoma L5178Y TK+/- cells (Varga-Kanizsai B. 2019) - negative for mutagenicity with and without metabolic activation.  

In vivo  

Mammalian erythrocyte micronucleus test in NMRI mice (Varga-Kanizsai B. 2019) - negative for the induction of micronuclei in bone marrow cells.  

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

There is no evidence for a mutagenic mode of action for dihexyl ether either in vitro or in vivo assays.