2-ethylhexyl nitrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxic potential of the registered substance has been excluded in a standard test battery: Ames test (OECD Guideline 471), chromosomal aberration assay (OECD Guideline 473) and mouse lymphoma assay (OECD Guideline 476). This was further confirmed in a mammalian cell transformation assay which was non-guideline and considered to be supporting data.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 17 April 2013 and 20 June 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 10 July 2012. Date of Signature: 30 November 2012

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Experiment 1: 0, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Experiment 2: 0, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

Vehicle / solvent:

Vehicle: dimethyl sulfoxide (DMSO)

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

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) - Experiment 1

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

METHODS OF APPLICATION: in agar (pre-incubation) – Experiment 2

- Pre-incubation period for bacterial strains: 10hrs
- Exposure duration: 48-72hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (in incubation with a selective agent): 20 minutes at 37 degrees C

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
-Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975),
Maron and Ames (1983) and Mortelmans and Zeiger (2000). All tester strain cultures should exhibit a characteristic number of spontaneous revertants per
plate in the vehicle and untreated controls. Combined historical negative and solvent control ranges for 2009 and 2010 are presented in Appendix 3. All
tester strain cultures should be in the range of 0.9e9 to 9e9 bacteria per ml. Diagnostic mutagens (positive control chemicals) must be included to
demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the
positive controls for 2009 and 2010 are presented in Appendix 3. There should be a minimum of four non-toxic test item dose levels. There should be no
evidence of excessive contamination.

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).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Standard Deviation
Dunnetts Linear Regression Analysis

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item caused a cytotoxic response to all of the Salmonella tester strains (except TA98 +S9-mix), initially from 50 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item caused a cytotoxic response to all of the Salmonella tester strains (except TA98 +S9-mix), initially from 50 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item caused a cytotoxic response to all of the Salmonella tester strains (except TA98 +S9-mix), initially from 50 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item caused a cytotoxic response to all of the Salmonella tester strains (except TA98 +S9-mix), initially from 50 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECFIC CONFOUNDING FACTORS:
- Precipitation: No precipitation was seen up to 5000 µg/plate.
- Purity: No correction required.
- Cytotoxicity: The test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the
frequency of revertant colonies of all of the Salmonella tester strains (except TA98 dosed in the presence of S9-mix), initially from 50 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence of S9-mix. The sensitivity of the
bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9 mix and experimental
methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum
recommended dose level of 5000 µg/plate.

STERILITY, VEHICLE AND POSITIVE CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The
amino acid supplemented top agar and S9 mix used in all experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for
concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.
A history profile of vehicle/untreated and positive control values (reference items) for 2011 and 2012 are presented in Appendix 2.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

RESULTS

Whilst the test item did not induce a reduction in the bacterial background lawns, substantial decreases in TA100 revertant colony frequency were noted from 500 µg/plate (absence of S9-mix) and at 5000 µg/plate (presence of S9-mix). No toxicity was noted to WP2uvrA. The test item formulation and S9‑mix used in this experiment were both shown to be sterile.

 

Mutation Test

Table 1: Spontaneous Mutation Rates (Concurrent Negative Control)

Experiment 1

 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
92		15		27		41		13
104	(98)	20	(15)	23	(23)	23	(30)	12	(13)
98		11		20		27		15

 

 

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
69		19		35		24		8
112	(96)	24	(21)	23	(27)	25	(23)	7	(7)
106		19		23		21		7

 

FOR TABLES OF RESULTS FOR MUTATION TEST: Please see attached in overall remarks, attachments

 

References:

Ames B N, McCann J and Yamasaki E (1975), Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Research, 31, 347-364.

Maron D M and Ames B N (1983), Revised Methods for the Salmonella mutagenicity test, Mutation Research, 113, 173 - 215.

Mortelmans K and Zeiger E (2000), The Ames Salmonella/microsome mutagenicity assay, Mutation Research, 455, 29-60.

Green M H L and Muriel W J (1976), Mutagen Testing Using TRP⁺ Reversion in Escherichia coli, Mutation Research, 38, 3- 32.

De Serres F J and Shelby M D (1979), Recommendations on data production and analysis using theSalmonella/microsome mutagenicity assay,Environmental Mutagenesis, 1, 87-92.

Mahon G A T et al (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report, 26-65.

Conclusions:

Interpretation of results (migrated information):
negative

The test item, 2-ethyl-hexyl nitrate, was considered to be non-mutagenic 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 (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, 2-ethyl-hexyl nitrate, using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range, fresh cultures of the bacterial strains and fresh test item formulations.

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve four non-toxic dose levels and the toxic limit of the test item. 

Results.The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive controls 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 test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of all of the Salmonella tester strains (except TA98 dosed in the presence of S9-mix), initially from 50 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrAat any test item dose level in either the absence or presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9‑mix and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate.

No significant 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 or exposure method. 

Conclusion.The test item,2 -ethyl-hexyl nitrate, was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 May 2005 to 03 October 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM culture medium

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1:
4-hour exposure without S9 mix, followed by 20-hour recovery: 0, 6.88, 13.75, 27.5, 55, 82.5, 110 µg/mL
4-hour exposure with S9 mix, followed by 20-hour recovery: 0, 13.75, 27.5, 55, 82.5, 110, 165 µg/mL

Experiment 2:
24-hour exposure without S9 mix: 0, 6.88, 13.75, 27.5, 55, 82.5, 110 µg/mL
4-hour exposure with S9 mix, followed by 20-hour recovery: 0, 13.75, 27.5, 55, 82.5, 110, 165 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 µg/mL)
STAIN (for cytogenetic assays): in 5% Gurrs Giemsa for 5 minutes

NUMBER OF CELLS EVALUATED: a total of 2000 lymphocyte cell nuclei were counted

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER OBSERVATION:
- polyploid cell frequency

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control group, either with or without a clear dose-relationship. For modest increases in aberration frequency, a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the maximum dose-level was 1760 µg/mL
- Precipitation: no precipitate was observed at the dose-levels of the main test (first and second experiment).

RANGE-FINDING/SCREENING STUDIES:
Concentrations: 0, 6.88, 13.75, 27.5, 55, 110, 220, 440, 880 and 1760 µg/mL
Precipitation: at and above 880 µg/mL in the 4-hour and 24-hour exposure tests without metabolic activation; at and above 440 µg/mL in the 4-hour exposure test with metabolic activation
Maximum doses with metaphase cells present: up to 55 and 110 µg/mL in the 4-hour exposure groups with and without metabolic activation respectively; 110 µg/mL in the 24-hour exposure group without S9 mix.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

All vehicle controls had frequencies of cells with aberrations within the range accepted for normal human lymphocytes.

All the positive control materials induces statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 March 2010 - 23 Sept 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Adequate coherence between data and conclusions. GLP and guideline compliant.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiments without S9 mix
Using a treatment volume of 0.5%, the selected dose-levels were as follows:
- 0.039, 0.078, 0.156, 0.313, 0.625 and 1.25 mM for the first experiment (3-hour treatment),
- 0.02, 0.039, 0.078, 0.156, 0.313, 0.625 mM for the second experiment (24 hour treatment).

Experiments with S9 mix
Using a treatment volume of 0.5%, the selected dose-levels were as follows:
- 0.156, 0.313, 0.625, 1.25, 2.5 and 5 mM for the first experiment,
- 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 mM for the second experiment.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: the test item was freely soluble in the vehicle (DMSO) at 350.46 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: (-S9) and cyclophosphamide (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.

Evaluation criteria:

IWGT recommendations were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the global evaluation
factor (GEF) of 126 x 10-6,
- a dose-related trend is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.
Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of
mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.

A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if:
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and
20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj.
RTG.

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Vehicle solubility: 350.46 mg/mL
- Precipitation: no precipitate was noted in the culture medium at the end of the treatment periods (3- or 24-hour treatments). A slight emulsion was observed at the end of the 3-hour treatment period at dose levels ≥ 2 mM.

RANGE-FINDING/SCREENING STUDIES: To assess the cytotoxicity of the test item, six dose-levels (one culture/dose-level) were tested both with and
without metabolic activation.
Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

ADDITIONAL INFORMATION:
Experiments without S9 mix
No precipitate was noted in culture medium at the end of the treatment periods (3- or 24-hour treatments).
Following the 3-hour treatment, a severe toxicity was induced at dose-levels ≥ 0.313 mM, as shown by a 89-96% decrease in Adj. RTG.
Following the 24-hour treatment, a moderate to severe toxicity was induced at dose levels ≥ 0.156 mM, as shown by a 44-100% decrease in Adj. RTG.
Following the 3-hour or 24-hour treatments, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.

Experiments with S9 mix
In the first experiment, a severe toxicity was induced at dose-levels ≥ 0.625 mM, as shown by a 82-100% decrease in Adj. RTG.
In the second experiment, a marked to severe toxicity was induced at dose-levels ≥ 0.625 mM, as shown by a 65-87% decrease in Adj. RTG.
No noteworthy increase in the mutation frequency was observed in either of the experiments.

Remarks on result:

other: strain/cell type: L5178Y

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item, 2-Ethylhexyl nitrate, did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat
metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item 2-Ethylhexyl nitrate to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK^+/-mouse lymphoma cells.

 

The study was performed according to international guidelines (OECD No. 476 and Commission Directive B 17) and in compliance with the principles of Good Laboratory Practice.

 

Methods

After a preliminary toxicity test, 2-Ethylhexyl nitrate was tested in two independent experiments, with and without a metabolic activation system

(S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 10⁵cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 10⁵cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a, 5% CO₂humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

Cytotoxicity wasmeasured by assessment of adjusted relative total growth (Adj. RTG), adjusted relative suspension growth (Adj. RSG) andcloning efficiency following the expression time (CE₂).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

The test item was diluted in dimethylsulfoxide (DMSO).

 

The dose-levels for the positive controls were as follows:

.           without S9 mix: methylmethane sulfonate (MMS), used at a final concentration of 25 µg/mL, (3-hour treatment) or 5 µg/mL (24-hour treatment),

.           with S9 mix: cyclophosphamide (CPA), used at a final concentration of 3 µg/mL.

 

Results

In this study, the cloning efficiencies (CE₂), the suspension growth and the mutation frequencies of the vehicle controls as well as the mutation frequencies of the positive controls were as specified in the acceptance criteria. The study was therefore considered to be valid.

In the culture medium, at the dose-level ofno precipitate was observed, and the pH and osmolarity values were equivalent to those of the vehicle control culture. Therefore,was selected as the highest dose-level to be tested in the preliminary toxicity test.

 

Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

Experiments without S9 mix

Using a treatment volume of 0.5%, the selected dose-levels were as follows:

.           0.039, 0.078, 0.156, 0.313, 0.625 andfor the first experiment (3-hour treatment),

.           0.02, 0.039, 0.078, 0.156, 0.313,for the second experiment (24‑hour treatment).

No precipitate was noted in culture medium at the end of the treatment periods (3- or 24-hour treatments).

 

Cytotoxicity

Following the 3-hour treatment, a severe toxicity was induced at dose-levels ≥ 0.313 mM, as shown by a 89-96% decrease in Adj. RTG.

Following the 24-hour treatment, a moderate to severe toxicity was induced at dose‑levels ≥ 0.156 mM, as shown by a 44-100% decrease in

Adj. RTG.

 

Mutagenicity

Following the 3-hour or 24-hour treatments, no noteworthy increase in the mutation frequency was noted in comparison to the vehicle control.

 

Experiments with S9 mix

Using a treatment volume of 0.5%, the selected dose-levels were as follows:

.           0.156, 0.313, 0.625, 1.25, 2.5 andfor the first experiment,

.           0.078, 0.156, 0.313, 0.625, 1.25 andfor the second experiment.

 

Cytotoxicity

In the first experiment, a severe toxicity was induced at dose-levels ≥ 0.625 mM, as shown by a 82-100% decrease in Adj. RTG.

In the second experiment, a marked to severe toxicity was induced at dose-levels ≥ 0.625 mM, as shown by a 65-87% decrease in Adj. RTG.

 

Mutagenicity

Nonoteworthy increase in the mutation frequencywas observed in either of the experiments.

Conclusion

The test item, 2-Ethylhexyl nitrate, did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on results from the available studies, 2 -EHN does not require classification for genetic toxicity according to Regulation (EC) No 1272/2008.