2-butoxyethyl benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are 3 key In Vitro GLP-studies available for 2-butoxyethyl benzoate. An OECD Guideline 471 (Bacterial Reverse Mutation Assay), an OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) and an OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test).

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 471, EPA OPPTS 870.5100, EU method B13/14 and in accordance with the Principles of Good Laboratory Practices (GLP)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Remarks:

Some minor exceptions were noted, however these did not have any impact on the outcome or results of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

same as above

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

same as above

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Remarks:

same as above

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures of tester strains were stored in Oxoid Nutrient Broth No. 2 in the test facility as frozen permanents in liquid nitrogen. Laboratory stocks were maintained on respective minimal glucose agar plates as master plates of each strain, for a maximum period of 2 months and refrigerated at 2 to 8 ºC.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: The growth requirements and the genetic identity of strains like histidine or tryptophan requirement, sensitivity to UV radiation, resistance of strains TA98, TA100 and WP2uvrA (pKM101) to ampicillin and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants after preparation of the master plates.
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S-9 homogenate was used as the metabolic activation system

Test concentrations with justification for top dose:

Initial Toxicity mutation Assay - G2 - 1.5 µg, G3 - 5 µg, G4 - 15 µg, G5 - 50 µg, G6 - 150 µg, G7 - 500 µg, G8 - 1500 µg and G9 - 5000 µg
Confirmatory Mutation Assay - G2 - 32 µg, G3 - 101 µg, G4 - 320 µg, G5 - 1013 µg, G6 - 3200 µg, G7 - 50 µg, G8 - 158 µg, G9 - 500 µg, G10 - 1580 µg and G11 - 5000 µg

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: recommended vehicle by various regulatory agencies

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Performance of the Assay -
Labeling - The petridishes were labelled to indicate the study number, strain number, treatment group, test phase and activation.
Number of Replicates - Two replicate plates were maintained for the initial toxicity-mutation assay and three replicate plates were maintained for the confirmatory mutation assay.
Plating Procedure - The initial toxicity-mutation, as well as the confirmatory mutation assay was conducted using the pre-incubation assay method at ambient temperature under yellow light.
A. Presence of metabolic activation -
a) 100 μl test dose/vehicle/appropriate positive control
b) 100 μl bacterial culture
c) 500 μl S-9 mix
B. Absence of metabolic activation
a) 100 μl test dose/vehicle/appropriate positive control
b) 100 μl bacterial culture
c) 500 μl of PBS
These test constituents were transferred into sterile test tubes and were kept in an incubator shaker for approximately 20 ± 2 minutes at 37 ± 1 ºC. After this period, 2 ml of soft agar containing histidine-biotin / tryptophan was added to each of the tubes and the constituents were overlaid onto VB agar plates. After the soft agar had set, the plates were incubated at 37 ± 1 °C for 67 hours.
After incubation, the revertant colonies in each plate were counted manually and the plates were examined for bacterial background lawn.
Viable Counts - The bacterial suspension of each tester strain was diluted up to 10-6 dilution in PBS. One hundred microliters (100 μl) from the 10-6 dilution of each tester strain was plated onto nutrient agar plates in triplicate. The plates were incubated at 37 ± 1 °C for 67 hours. After incubation, the number of colonies in each plate were manually counted and expressed as the number of colony forming units per ml of the bacterial suspension

Evaluation criteria:

The conditions necessary for determining a positive result were - there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test substance either in the absence or presence of the metabolic activation system.
For strains TA98, TA1535, and TA1537 - Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
For strain TA100 and WP2uvrA (pKM101) - Data sets were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) were not considered as positive.

Statistics:

Standard statistical methods were employed

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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

Positive controls validity:

valid

Additional information on results:

S-9 Homogenate
a) Sterility Check - The S-9 homogenate was found to be sterile.
b) Metabolic Activation - The S-9 homogenate was found to be active.
c) Protein Content - The protein content of the S-9 homogenate was 27.5 mg/mL.
Genotypic Characterization - Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 demonstrated the requirement of histidine amino acid for their growth. Escherichia coli strain WP2uvrA (pKM101) demonstrated the requirement of tryptophan amino acid for its growth. Ampicillin resistance was demonstrated by the strains TA98, TA100 and WP2uvrA (pKM101) which carry R-factor plasmids. The presence of characteristic mutations like the rfa mutation was demonstrated by all the Salmonella typhimurium strains by their sensitivity to crystal violet. The uvrA mutation in the Escherichia coli strain and the uvrB mutation in the Salmonella typhimurium strains were demonstrated through their sensitivity to ultraviolet light.
Finally, all these tester strains produced spontaneous revertant colonies which were within the frequency ranges of the test facility’s historical control data.
Viable Counts of the Overnight Culture - Viable counts of all the tester strains were within the required range of 1-2x109 CFU/mL for the initial toxicity-mutation as well as for the confirmatory mutation assay.
Stability Analysis of Dose Formulation Samples - The test substance was stable in DMSO at 15 and 50000 μg/mL after 4 and 24 hours.
Concentration Analysis of Dose Formulations - The results of the concentration analysis for the initial toxicity-mutation assay indicated that the actual mean concentrations of the analyzed dose levels were between 89.5 and 105 % of their respective nominal target concentrations confirming that the concentration of the test substance was within acceptable limits (85 to 115 % of nominal concentrations and an RSD of < 10 %), except for the dose 15 μg/mL, which was higher than the acceptable limit (117 %), no additional testing was conducted to investigate this finding. This indicates that the regulatory-required top dose level (5000 μg/plate) was achieved. No test substance was detected in the vehicle control.
The results of the concentration analysis for the confirmatory mutation assay indicated that the actual mean concentrations of the analyzed dose levels were between 101 and 109 % of their respective nominal target concentrations confirming that the concentration of the test substance was within acceptable limits (85 to 115 % of nominal concentrations and an RSD of < 10 %), except for the dose 500 μg/mL, which was higher than the acceptable limit (180 %), no additional testing was conducted to investigate this finding. This indicated that the regulatory-required top dose level (5000 μg/plate) was achieved. No test substance was detected in the vehicle control.
Initial Toxicity-Mutation Assay - The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains The test substance did not precipitate on the basal agar plates at any of the tested doses
but showed differential toxicity profile. No toxicity was observed in any of the tester strains up to 500 µg/plate, either in the presence or absence of metabolic activation when compared to the vehicle control. However, for TA98, TA100, TA1535 and TA1537, there was a slight reduction in the intensity of bacterial background lawn at 1500 µg/plate and moderate reduction at 5000 µg/plate. For WP2uvrA (pKM101), there was a slight reduction in the intensity of bacterial background lawn at 1500 as well as at 5000 µg/plate. There was no positive mutagenic response observed in any of the strains in any of the tested doses either in the presence or in the absence of metabolic activation.
Positive control chemicals tested simultaneously produced more than a 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.
Confirmatory Mutation Assay - The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains. The test substance did not precipitate on the basal agar plates at any of the tested
doses. No toxicity was observed in the tester strains TA98, TA100, TA1535 and TA1537 up to 320 µg/plate and up to 500 µg/plate in the strain WP2uvrA (pKM101), either in the presence or absence of metabolic activation when compared to the vehicle control. However, for TA98, TA100, TA1535 and TA1537, there was a slight reduction in the intensity of bacterial background lawn at 1013 and 3200 µg/plate. For WP2uvrA (pKM101), there was a slight reduction in the intensity of bacterial background lawn at 1580 as well as at 5000 µg/plate. There was no positive mutagenic response observed in any of the strains in any of the tested doses either in the presence or in the absence of metabolic activation.
Positive control chemicals tested simultaneously produced more than a 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.
Sterility Controls - The most concentrated test substance dilution, the Sham (PBS) and S9 mixes were found to be sterile.
Both the Salmonella typhimurium and Escherichia coli tester strains were found to be reliable and responsive to the different genotypic characterization tests, e.g., amino acid requirement, rfa mutation, uvr mutation and the R-factor plasmids. Similarly, the spontaneous revertant counts of the vehicle control groups of these tester strains were within the ranges of the test facility’s historical control data.
The positive controls produced a more than 3-fold increase in the mean numbers of revertant colonies when compared to the respective vehicle controls, demonstrating the sensitivity of the assay procedure.
The test substance, Butyl CELLOSOLVE™ Benzoate tested at doses up to 5000 μg/plate, did not show any positive mutagenic response in any of the tester strains in any of the tested doses either in the presence or absence of metabolic activation when compared to the respective vehicle control plates.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

None

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this assay, it is concluded that the test substance, 2-butoxyethyl benzoate was negative (non-mutagenic) in this Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay.

Executive summary:

In a confirmatory mutation assay, 2 -butoxyethyl benzoate was exposed in triplicate at 32, 101, 320, 1013 and 3200 µg/plate test doses with TA98, TA100, TA1535 and TA1537 and at 50, 158, 500, 1580 and 5000 µg/plate test doses with WP2uvrA (pKM101) along with the vehicle and appropriate positive controls. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains. The test substance did not precipitate on the basal agar plates at any of the tested doses. No toxicity was observed in the tester strains TA98, TA100, TA1535 and TA1537 up to 320 µg/plate and up to 500µg/plate in the strain WP2uvrA (pKM101), either in the presence or absence of metabolic activation when compared to the vehicle control. However, for TA98, TA100, TA1535 and TA1537, there was a slight reduction in the intensity of bacterial background lawn at 1013 and 3200 µg/plate. For WP2uvrA (pKM101), there was a slight reduction in the intensity of bacterial background lawn at 1580 as well as at 5000 µg/plate. There was no positive mutagenic response observed in any of the strains in any of the tested doses either in the presence or in the absence of metabolic activation. In this study, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay. The results of concentration analysis of dose formulations from the initial and confirmatory mutation assays confirmed that the regulatory-required top dose level (3200 and 5000 μg/plate) was achieved in both assays and the results support the validity of the study conclusion. Under the conditions of the current study, the test substance, 2 -butoxyethyl benzoate was negative (nonmutagenic) in thisSalmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2, 2015 to November 12, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Ministry of Economy, Trade and Industry - IV. Mutagenicity Test, Chromosome Aberration Test Using Cultured Mammalian Cells

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Chromosomal aberration assay utilizing rat lymphocytes.

Specific details on test material used for the study:

Test Material Name: 2-Butoxyethyl benzoate
Chemical Name: 2-Butoxyethanol benzoate
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Midland, Michigan (Lot # 201303443-19).
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area (corrected for water) by gas chromatography with identification by nuclear magnetic resonance and gas
chromatography mass spectrometry (Gobbi, 2014).
Test Material Stability Under Storage Conditions: The test material was determined to have two years of stability under ambient storage conditions (Wachowicz el al., 2015).

Target gene:

Chromosomal aberration assay utilizing rat lymphocytes.

Species / strain / cell type:

lymphocytes: Rat

Details on mammalian cell type (if applicable):

Species and Sex: Rats (Male)
Strain and Justification: Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location: Charles River (Kingston, New York)
Age at Study Start: Approximately 10 weeks

Lymphocyte Cultures:
Blood samples were collected by cardiac puncture, following euthanasia with carbon dioxide. In the assays, blood samples from individual rats were pooled and whole blood cultures were set up in RPMI 1640 medium (with 25 mM HEPES, GIBCO, Grand Island,
New York) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO),
antibiotics and antimycotics (Fungizone 0.25 μg/ml; penicillin G, 100 u/ml; and
streptomycin sulfate, 0.1 mg/ml; GIBCO), 30 μg/ml PHA-P (HA16, Remel Europe Ltd.,
Dartford, England), and an additional 2 mM L-glutamine (GIBCO). Cultures were
initiated by inoculating approximately 0.5 ml of whole blood/5 ml of culture medium.
Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks
and incubated at 37°C.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate prepared from Aroclor 1254-treated (500 mg/kg body weight) male Crl:CD(SD) rats.

Test concentrations with justification for top dose:

Cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 425.0 μg 2-Butoxyethyl benzoate per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium.

Vehicle / solvent:

The test material was found to be soluble in dimethyl sulfoxide (DMSO; CAS No. 67-68-5) up to 339.14 mg/ml. Therefore DMSO was selected as the solvent to dissolve the test material and was used as the vehicle control treatment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other:

Details on test system and experimental conditions:

Lymphocyte Cultures:
Blood samples were collected by cardiac puncture, following euthanasia with carbon dioxide. In the assays, blood samples from individual rats were pooled and whole blood cultures were set up in RPMI 1640 medium (with 25 mM HEPES, GIBCO, Grand Island, New York) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO), antibiotics and antimycotics (Fungizone 0.25 μg/ml; penicillin G, 100 u/ml; and streptomycin sulfate, 0.1 mg/ml; GIBCO), 30 μg/ml PHA-P (HA16, Remel Europe Ltd., Dartford, England), and an additional 2 mM L-glutamine (GIBCO). Cultures were initiated by inoculating approximately 0.5 ml of whole blood/5 ml of culture medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C.

In Vitro Metabolic Activation System:
S9 liver homogenate prepared from Aroclor 1254-treated (500 mg/kg body weight) male Crl:CD(SD) rats was purchased from Molecular Toxicology Inc., Boone, North Carolina, and stored at -100°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a "mix" (O'Neill et al., 1982). The mix consisted of 10 mM MgCl2·6H2O (Sigma), 5 mM glucose-6-phosphate (Sigma), 4 mM nicotinamide adenine dinucleotide phosphate (Sigma), 10 mM CaCl2 (Sigma), 30 mM KCl (Sigma), and 50 mM sodium phosphate (pH 8.0, Sigma). The reconstituted mix was added to the culture medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the culture medium was 1/5 of the concentrations stated above.

Preparation of the Treatment Solution and Administration of the Test Material:
All test material solutions were prepared fresh on the day of treatment and used within two hours of preparation. The test material was dissolved in DMSO and further diluted (1: 100) in treatment medium. This technique has been shown to be an effective method for detecting various in vitro clastogens in this test system. All dosing units were expressed in μg/ml. MMC was dissolved in RPMI 1640 with HEPES and antibiotics, and CP was dissolved in distilled water.

Analytical Verification of Dosing Solutions:
The selected concentrations of the test material in the stock dosing solutions used for treatment in Assay A1 were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by high performance liquid chromatography with ultraviolet detection (HPLC/UV). Homogeneity analysis was not conducted as the test material was not administered as a suspension.

Identification of the Test System:
All test cultures were identified using self adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

Evaluation criteria:

Evaluation Criteria:
For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the vehicle controls. The aberration frequency in the vehicle control should be within reasonable limits of the laboratory historical values. A test chemical will be considered positive in this assay if it induces a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells is outside the recent historical vehicle control range.

Statistics:

Statistical Analysis:
The proportions of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two-way contingency table was constructed to analyze the frequencies of aberrant cells. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the global hypothesis of no difference in the average number of cells with aberrations among the dose groups (Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If this statistic was found to be significant at alpha = 0.05, versus a one-sided increasing alternative, pairwise tests (i.e. control vs. treatment) were performed at each dose level and evaluated at alpha = 0.05, again versus a one-sided alternative. If any of the pairwise tests were significant, a test for linear trend of increasing number of cells with aberrations with increasing dose was performed (Armitage, 1971). Polyploid cells were analyzed by the Fisher Exact probability test (Siegel, 1956). The number of polyploid cells were pooled across replicates for the analysis and evaluated at alpha = 0.05. The data was analyzed separately based on the presence or absence of S9 and based on the exposure time.

Key result

Species / strain:

lymphocytes: Rat

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No significant increases in the incidence of polyploid cells or frequency of cells with aberrations in the test material treated cultures (26.6, 106.3, and 425.0 μg/ml) as compared to the vehicle control.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion). The test material precipitated in the treatment medium at the top two concentrations (i.e., 212.5 and 425.0 μg/ml).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Rat

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No significant increases in the incidence of polyploid cells or frequency of cells with aberrations in the test material treated cultures (90.0, 130.0, and 190.0 μg/ml) as compared to the vehicle control.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion). The test material precipitated in the treatment medium at the top two concentrations (i.e. 190.0 and 212.5 μg/ml).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Rat

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No significant increases in the incidence of polyploid cells or frequency of cells with aberrations in the test material treated cultures (90.0, 130.0, and 170.0 μg/ml) as compared to the vehicle control.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion). The test material precipitated in the treatment medium at the top two concentrations (i.e. 190.0 and 212.5 μg/ml).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

pH and Osmolality:
The pH and osmolality of treatment medium containing approximately 3391.4 μg/ml of the test material and medium containing 1% DMSO were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts), respectively. There was no appreciable change in either the pH or osmolality at this concentration as compared to the culture medium with solvent alone (culture medium with the test material, pH = 7.40, osmolality = 398 mOsm/kg H2O; culture medium with 1% DMSO, pH = 7.38, osmolality = 441 mOsm/kg H2O).

Remarks on result:

other: Assay A1 4-Hour Treatment

Assay A1:

In the initial assay, cultures were treated with the test material in the absence and presence of S9 activation for 4 hours at concentrations of 0 (vehicle control), 6.6, 13.3, 26.6, 53.1, 106.3, 212.5, and 425.0 μg/ml (Tables 1 and 2). Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 3.3 μg/ml (Table 3). The test material precipitated in the treatment medium at the top two concentrations (i.e., 212.5 and 425.0 μg/ml) in all treatment conditions. Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 97.7 to 101.1% of the target and verified that concentrations used for treatment were within an acceptable range.

Short Treatment:

In the absence of S9, excessive toxicity was observed at the highest two concentrations (212.5 and 425.0 μg/ml) as indicated by no observable mitotic figures. At the remaining concentrations substantial to no toxicity was observed with relative mitotic indices ranging from 49.7 to 97.0 as compared to the vehicle control value. Based upon these results, this portion of the assay was repeated as Assay B1, due to not achieving the recommended level of cytotoxicity (i.e. 60 +10% reduction in mitotic index) and an inconsistent dose-response.

In the presence of S9, cultures displayed moderate toxicity with relative mitotic indicies ranging from 75.9 to 106.4 as compared to the vehicle control value. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 26.6, 106.3, and 425.0 μg/ml (precipitating concentration) were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy in the presence of S9 activation. Among the cultures treated with positive control chemicals for 4 hours, 2 μg/ml of CP was selected for evaluation of aberrations in the presence of S9.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures (4-hour treatment, with S9) as compared to the vehicle control values. In the presence of S9, cultures treated with the test material at concentrations of 26.6, 106.3, and 425.0 μg/ml had aberrant cell frequencies of 0.5, 0.5, and 0.5%, respectively as compared to the vehicle control value of 1.0%. Statistical analyses of these data did not identify significant difference between the vehicle control and any of the test material-treated cultures

with S9 activation and all values were within the laboratory historical background range. Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in CP (+S9, 4 hour treatment) treated cultures were 40.0%. A second assay with treatment of cultures for 4 hours in the presence of S9 was not considered necessary since the results of the initial test yielded clearly negative results.

Continuous Treatment:

Cultures treated continuously for 24 hours in the absence of S9 activation displayed excessive toxicity as observed by no mitotic figures at the two highest concentrations (212.5 and 425.0 μg/ml). Little to no toxicity was observed at the remaining cultures with relative mitotic indices ranging from 72.5 to 97.7% as compared to the vehicle control value. Based upon these results, this portion of the assay was repeated as Assay B1, due to not achieving the recommended level of cytotoxicity (i.e. 60 +10% reduction in mitotic index).

Assay B1 – Repeat Cytogenetic Assay:

In a repeat assay in the absence of S9, cultures were treated with target concentrations of 0 (vehicle control), 10.0, 90.0, 110.0, 130.0, 150.0, 170.0, 190.0, and 212.5 μg/ml for 4 and 24 hours. The test material precipitated in the treatment medium at the top two concentrations (i.e. 190.0 and 212.5 μg/ml).

Short Treatment:

In the absence of S9, substantial to no toxicity was observed with relative mitotic indices ranging from 11.1 to 98.7%. Based upon these results, cultures treated with 0 (vehicle control), 90.0, 130.0, and 190.0 μg/ml in the absence of S9 were selected for determining the chromosomal aberration frequencies and incidence of polyploidy. The cultures treated with the positive control chemical, 0.5 μg/ml MMC were also evaluated for chromosome aberrations.

There were no significant increases in the incidence of polyploidy cells in test material treated cultures as compared to the vehicle control values. In the absences of S9, cultures treated with the test material at concentrations 90.0, 130.0, and 190.0 μg/ml had aberrant cell frequencies of 0.5, 0.0, and 0.5%, respectively, as compared to the vehicle control values of 0.5%. Statistical analysis of these data did not identify significant differences between the vehicle control and any of the test material-treated cultures without S9 activation. The frequencies of aberrant cells observed in the vehicle control and test material treated cultures were within the laboratory historical background range. Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical (0.5 MMC; 26%).

Continuous Treatment:

In the absence of S9, cultures displayed excessive toxicity at the top two concentrations (190.0 and 212.5 μg/ml) with the remaining cultures having relative mitotic indices ranging from 23.8 to 81.1%. Based upon these results, targeted concentrations of 0 (vehicle control), 90.0, 130.0, and 170.0 μg/ml were selected for evaluation of chromosomal aberrations and the incidence of polyploidy. Among the cultures treated with the positive control chemical, 0.075 μg/ml of MMC was selected for the evaluation of aberrations.

There were no significant increases in the incidence of polyploid cells in test material treated cultures as compared to the vehicle control values. In the absence of S9, cultures treated with the test material at concentrations of 90.0, 130.0, and 170.0 μg/ml had aberrant cell frequencies of 0.0, 0.0, and 0.0%, respectively, as compared to the vehicle control values of 0.5%. Statistical analysis of these data did not identify significant difference between the vehicle control and any of the test material-treated cultures without S9 activation and all values were within the laboratory historical background range. Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical (0.075 μg/ml MMC; 19.0%).

Conclusions:

It was concluded that under the experimental conditions used, 2-Butoxyethyl benzoate was negative in the in vitro chromosomal aberration test.

Executive summary:

2-Butoxyethyl benzoate was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 425.0 μg 2-Butoxyethyl benzoate per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The duration of treatment was 4 or 24 hours without S9 and 4 hours with S9. The analytically determined concentrations of 2-Butoxyethyl benzoate in the dose preparations ranged from 97.7 to 101.1% of the targeted values. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon solubility of the test material and the mitotic index values. In this study cultures treated for 4 hours with targeted concentrations of 0 (vehicle control), 26.6, 106.3, and 425.0 μg/ml in the presence of S9, 0 (vehicle control), 90.0, 130.0, and 190.0 μg/ml in the absence of S9, and cultures treated for 24 hours with 0 (vehicle control), 90.0, 130.0 and 170.0 μg/ml in the absence of S9 were analyzed for chromosomal aberrations.

There were no significant increases in the frequency of cells with aberrations administered 2-Butoxyethyl benzoate in either the absence or presence of S9 activation. In addition, the frequencies of aberrant cells observed in the test material treated cultures were within the laboratory historical background range. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of aberrant cells in all assays. Based upon these results, 2-Butoxyethyl benzoate was considered to be negative in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 8, 2015 to November 13, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: (CHO/HGPRT) forward gene mutation assay

Specific details on test material used for the study:

Test Material Name: 2-Butoxyethyl benzoate
Chemical Name: 2-Butoxyethanol benzoate
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Midland, Michigan (Lot # 201303443-19).
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area (corrected for water) by gas chromatography with identification by nuclear magnetic resonance and gas chromatography mass spectrometry (Gobbi, 2014).
Test Material Stability Under Storage Conditions: The test material was determined to have two years of stability under ambient storage conditions (Wachowicz, et. al., 2015).

Target gene:

The gene for Hgprt is located on the mammalian X-chromosome.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Indicator Cells and Justification of Their Use:
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, Tennessee, was used in this study. The CHO-K1-BH4 cell line was selected as the test system for this study because it is sensitive to mutagens, has a low background mutant frequency, and is readily available. Stock cultures were stored at about -80°C or below. The cultures were periodically checked for mycoplasma contamination (American Type Culture Collection, Manassas, Virginia). The cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of approximately 5% CO2 in air at 37°C in a humidified incubator.

Media:
The cells were routinely maintained in Ham's F-12 nutrient mix (GIBCO, Grand Island, New York) supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum (GIBCO), antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 0.25 μg/ml; GIBCO), and an additional 2 mM L-glutamine (GIBCO). The selection medium used for the detection of Hgprt-mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 μM 6-thioguanine (GIBCO), 5% serum, and the above-mentioned antibiotics.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats.

Test concentrations with justification for top dose:

The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 26.6 to 425 μg/ml in the absence of S9 and in the presence of S9. The highest concentration was based on limit of solubility of the test material in the treatment medium.

Vehicle / solvent:

The test material was found to be soluble in dimethyl sulfoxide (DMSO; CAS No. 67-68-5) up to 339.14 mg/ml, therefore, DMSO was selected as the solvent used to dissolve the test material and was used as the vehicle control treatment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: The positive control for assays performed with S9 (activation system) was 20-methylcholanthrene (20-MCA, CAS No. 56-49-5) at concentrations of 4 and 8 μg/ml.

Details on test system and experimental conditions:

Controls:
The test material was found to be soluble in dimethyl sulfoxide (DMSO; CAS No. 67-68-5) up to 339.14 mg/ml, therefore, DMSO was selected as the solvent used to dissolve the test material and was used as the vehicle control treatment. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was used as the positive control for the non-activation system (without S9 factor) at a final concentration of 621 μg/ml. The positive control for assays performed with S9 (activation system) was 20-methylcholanthrene (20-MCA, CAS No. 56-49-5) at concentrations of 4 and 8 μg/ml. The dose levels of EMS and 20-MCA were based upon our unpublished findings.

Preparation of the Treatment Solution and Administration of the Test Material:
All test material solutions were prepared fresh on the day of treatment and used within three hours of preparation. The test material was first dissolved in DMSO and further diluted (1: 100) in treatment medium to obtain the desired concentrations as recommended in the test guidelines. This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et. al., 1981). EMS was dissolved in treatment medium. 20-MCA was dissolved first in dimethyl sulfoxide and further diluted in the culture medium. All dosing units were expressed in μg/ml.

Treatment Procedure:
Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 x 10^6 cells/T-75 flask approximately 24 hours prior to treatment. At the time of treatment, the culture medium was replaced with serum-free medium, S9 mix (when applicable), and the test chemical, the vehicle control, or the positive control chemical. The cells were treated for approximately 4 hours at 37°C and the exposure was terminated by washing the cells with phosphate buffered saline (Ca++and Mg++ free).

Identification of the Test System:
All test cultures were identified using self adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

Analytical Verification of Dosing Solutions:
The selected concentrations of the test material in the stock dosing solutions used for treatment in Assay B1 were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by high performance liquid chromatography with ultraviolet detection (HPLC/UV). Homogeneity analysis was not conducted as the test material was not administered as a suspension.

In Vitro Metabolic Activation:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats were purchased from a commercial source and stored at approximately -80°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a “mix” (O’Neill et al., 1982). The S9 mix consisted of the following co-factors: 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the culture medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the culture medium is 1/5th of the concentrations stated above.

Evaluation criteria:

Evaluation Criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the vehicle controls. An additional criterion was that the mutant frequency in the vehicle controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose-related, reproducible increase in mutant frequency. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the vehicle controls.

Statistics:

Statistical Analysis:
The frequency of mutants per 10^6 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutant frequency variance. The actual plate counts are assumed to follow a Poisson distribution, therefore, the mean plate count was used as an estimate of variance (Kirkland, 1989).
If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

other: The highest concentrations (212.5 and 425 μg/ml) precipitated in the treatment medium. Treated cultures showed relative cell survival (RCS) values (%) ranging from 83.6 to 110.9 in the absence of S9 and 95.9 to 126.1 in the presence of S9.

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

other: The highest concentrations (212.5 and 425 μg/ml) precipitated in the treatment medium. Without S9, there was little to no toxicity (RCS values from 85.6 to 129.7%). With S9, there was moderate to no toxicity (RCS values from 41.4 to 122.4%).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

other: The highest concentrations (212.5, and 425 μg/ml) precipitated in the treatment medium. There was ittle to no toxicity observed, as indicated by the RCS values (%), in the absence and presence of S9 (51.9 to 108.7 and 72.1 to 108.7 respectively).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

pH and Osmolality
The pH and osmolality of treatment medium containing approximately 3391.4 μg/ml of the test material and medium containing 1% DMSO were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays (Thilagar et al., 1984; Galloway et al., 1985; Cifone, 1985). There was no appreciable change in either the pH or osmolality at this concentration as compared to the culture medium with solvent alone (culture medium with the test material, pH = 7.36, osmolality = 416 mOsm/kgH2O; culture medium with 1% DMSO, pH = 7.36, osmolality = 468 mOsm/kgH2O).

Remarks on result:

other: Results for Assay A1 - Preliminary Toxicity Assay

Assay A1 – Preliminary Toxicity Assay:

In a preliminary toxicity assay, the test material was tested at concentrations of 0 (vehicle control), 1.7, 3.3, 6.6, 13.3, 26.6, 53.1, 106.3, 212.5 and 425 μg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest two concentrations (212.5 and 425 μg/ml) precipitated in the treatment medium. The treated cultures in the absence and presence of S9 activation showed little to no toxicity with the relative cell survival (RCS) values ranging from 83.6 to 110.9 in the absence of S9 and 95.9 to 126.1 in the presence of S9. Based upon the results of this assay, target concentrations of 0 (vehicle control), 26.6, 53.1, 106.3, 212.5, and 425 μg/ml of the test material were selected for the initial gene mutation assay in the absence of S9 and presence of S9.

Assay B1 – Initial Mutagenicity Assay:

In the initial mutagenicity assay (Assay B1), the highest two concentrations (212.5 and 425 μg/ml) precipitated in the treatment medium. In the absence of S9, little to no toxicity was observed with RCS values ranging from 85.6 to 129.7%. In the presence of S9, moderate to no toxicity was observed with RCS values ranging from 41.4 to 122.4%. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 were not significantly different from the concurrent vehicle control values. All mutant frequencies were within a reasonable range of historical background values.

Assay C1 – Confirmatory Mutagenicity Assay:

Based upon the previous results, targeted concentrations of 0 (vehicle control), 26.6, 53.1, 106.3, 212.5, and 425 μg/ml in the absence and presence of S9 were selected for the confirmatory gene mutation assay. In this assay, the highest two concentrations precipitated in the treatment medium. There was little to no toxicity observed, as indicated by the RCS values, in the absence of S9 (51.9 to 108.7%). In the presence of S9, RCS values indicated little to no toxicity with RCS values ranging from 72.1 to 108.7. The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values and were within the range of the laboratory historical background.

In both the initial and confirmatory mutagenicity assays, the positive control chemicals induced significant increases in mutation frequencies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations.

The analytically observed concentrations of the test material in the stock dosing solutions in Assay B1 ranged from 97.7 to 101.3% of target and verified that concentrations used for treatment were within acceptable range.

Conclusions:

The results of the in vitro Chinese Hamster Ovary cell/hypoxanthine-guaninephosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay with 2-
Butoxyethyl benzoate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

Executive summary:

2-Butoxyethyl benzoate was evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 26.6 to 425 μg/ml in the absence of S9 and in the presence of S9. The highest concentration was based on limit of solubility of the test material in the treatment medium. The analytically determined concentrations of 2-Butoxyethyl benzoate in the dose preparations ranged from 97.7 to 101.3%. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Vehicle control cultures were treated with the solvent used to dissolve the test material (i.e. dimethyl sulfoxide).

There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the vehicle control cultures in either the absence or presence of S9. The results of the CHO/HGPRT forward gene mutation assay with 2-Butoxyethyl benzoate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There is 1 key In Vivo GLP-study available for 2-butoxyethyl benzoate. An OECD Guideline 474 (Mammailan Erythrocyte Micronucleus Test).

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:

April 30, 2015 to December 3, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Mouse peripheral blood micronucleus test

Specific details on test material used for the study:

Test Material Name: 2-Butoxyethyl benzoate
Chemical Name: 2-Butoxyethanol benzoate
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Midland, Michigan (Lot # 201303443-19).
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area (corrected for water) by gas chromatography with identification by nuclear magnetic resonance and gas chromatography mass spectrometry (Gobbi, 2014).
Test Material Stability Under Storage Conditions: The test material was determined to have two years of stability under ambient storage conditions (Wachowicz, et al., 2015).

Species:

mouse

Strain:

other: Outbred Crl:CD1(ICR)

Details on species / strain selection:

Criteria for Selecting the Strain:
1. Availability of historical negative control data
2. Suitability for utilization in the MNT
3. General suitability for toxicity testing

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex: Male and female mice were used for the RFT. Only males were used for the MNT due to the results of the RFT, which showed no apparent difference in the toxicity between the sexes.
Strain: Outbred Crl:CD1(ICR) mice
Criteria for Selecting the Strain:
1. Availability of historical negative control data
2. Suitability for utilization in the MNT
3. General suitability for toxicity testing
Supplier and Location: Charles River (Kingston, New York)
Age at Study Start: Approximately 8-10 weeks

Physical Acclimation:
During the acclimation period each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

Housing:
After assignment, animals were housed one per cage in stainless steel cages. Cages had wire mesh floors and were suspended above absorbent paper. Non-woven gauze was placed in the cages to provide a cushion from the flooring for the rodents' feet. The gauze also provided environmental enrichment. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 40-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Randomization and Identification:
Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water:
Feed and municipal water were provided ad libitum. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained in the study file.

Animal Welfare:
In accordance with the U.S. Department of Agriculture Animal Welfare Regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study were Genetic Tox 01, Blood Collection 01, Humane Endpoints 01, and Animal ID 01.

Route of administration:

oral: gavage

Vehicle:

The vehicle used to mix the test material (corn oil; CAS number 8001-30-7), served as the negative control.

Details on exposure:

Dose Levels:
The dosing solutions of the test material were prepared and used fresh on each of the two consecutive days of administration. A frozen stock solution of CP dissolved in distilled water (thawed and brought to room temperature prior to use) served as the positive control. The vehicle used to mix the test material (corn oil; CAS number 8001-30-7), served as the negative control. The concentrations of the test material in the dosing solutions used for the first day of dosing were verified using high performance liquid chromatography with ultraviolet detection (HPLC/UV).

Animal Dosing:
The test material, the vehicle (negative control), and CP (positive control) were administered by oral gavage. Oral gavage is one of the generally accepted routes of administering the test material in this assay system. CP when administered by oral gavage has been shown to induce micronuclei in polychromatic erythrocytes of Crl:CD1(ICR) mice (De Boeck et al., 2005; Gollapudi et al., 1986). Dosing solutions were administered in aliquots of 10 ml/kg bodyweight (bw).

Duration of treatment / exposure:

In the Micronucleus Test (MNT), groups of male mice were administered 0, 375, 750, or 1500 mg/kg bw/day of the test material on two consecutive days. CP was administered only once at a dose level of 40 mg/kg bw. The dose levels were based on the results of the RFT.

Frequency of treatment:

Once per day.

Post exposure period:

48 hours

Dose / conc.:

375 mg/kg bw/day

Dose / conc.:

750 mg/kg bw/day

Dose / conc.:

1 500 mg/kg bw/day

No. of animals per sex per dose:

There were six mice/dose except in the 1500 mg/kg bw/day group where an additional group of two mice were dosed as possible replacements in the event of deaths occurring among the treated animals of this group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive Control Chemical:
Chemical Name: Cyclophosphamide monohydrate (CP)
CAS Number: 6055-19-2
Source: Sigma, St. Louis, Missouri

CP was administered only once at a dose level of 40 mg/kg bw.

Tissues and cell types examined:

Cells Examined:
Approximately 5,000 RETs were analyzed per blood sample. The number of normochromatic erythrocytes (NCE), MN-NCE, RET, and MN-RET were recorded for
each sample and the frequency of MN-RET was determined to provide an indication of genotoxic potential. The frequency of RETs relative to total erythrocytes was determined to provide an indication of perturbations in hematopoietic activity indicative of cell toxicity. For each of the treatment groups, a mean and standard deviation was calculated to describe the frequency of RET and MN-RET observed. The analyses were conducted utilizing a flow cytometer (Beckman Coulter Gallios flow cytometer).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose levels were based on the results of the RFT.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Rationale for Selection of Sampling Intervals for Micronucleus Test (MNT):
It takes approximately 48 hours for the treated erythroblasts to undergo one posttreatment division and mature into reticulocytes and reach a peak incidence in the peripheral blood (CSGMT, 1992). However, if the treatment induces cell cycle delay, or if there is a delayed absorption and metabolism of the test material, a longer sampling time (such as 72 hours) may be needed to recover the damage induced in the erythroblasts. The sampling time used in this study provided an opportunity to recover any delayed effect (i.e., effect induced by the first treatment) as well as a more immediate effect (i.e., effect induced by the second treatment) in the same treated animals.

Peripheral Blood:
Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry (FCM; Beckman Coulter Gallios) with traditional blood smears prepared as a backup. Samples were prepared and analyzed per instructions in the Mouse MicroFlow Micronucleus Analysis Kit Manual (Litron Laboratories, Rochester, New York). At the end of the specified interval following treatment, a peripheral blood sample was collected from the orbital sinus of all surviving animals into anticoagulant solution following anesthesia with isoflurane. Briefly, the blood samples were fixed in ultracold (-70 to -80°C) methanol within five hours of collection. All fixed blood samples were stored at approximately -80°C until the time of analysis. Fixed blood samples were washed with a cold, buffered salt solution and isolated by centrifugation. The resulting cell pellets were stored at 4°C until staining. Blood samples were ultimately incubated with RNase to degrade the high levels of RNA present in the reticulocytes (RET) and a fluorescently labeled antibody to the transferrin receptor (anti-CD71-FITC) to specifically identify the RET. A propidium iodide solution was added to each sample immediately before FCM analysis to stain the DNA, including that of micronuclei. In this system, the sample was moved at a high velocity past a laser set to provide 488 nm excitation. The fluorescent wavelengths emitted by each cell were collected by photomultiplier tubes. Using the previously described staining procedure, the propidium iodide-stained DNA of the micronuclei emitted a red fluorescence and the anti-CD71-FITC antibody emitted a high green fluorescent signal permitting differentiation between cells with and without micronuclei. In addition to obtaining fluorescent profiles, FCM simultaneously provided cell size information by determining the light scatter properties of each cell or combination of cells.

DETAILS OF SLIDE PREPARATION:
Duplicate cell smears were prepared and stored to serve as backups in the event that the flow cytometric analysis was not possible. Blood was collected into a microtainer tube coated with EDTA (Becton Dickinson, Franklin Lakes, New Jersey). Wedge smears were prepared, fixed in methanol, and stored at room temperature.

METHOD OF ANALYSIS:
Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry (FCM; Beckman Coulter Gallios) with traditional blood smears prepared as a backup. Samples were prepared and analyzed per instructions in the Mouse MicroFlow Micronucleus Analysis Kit Manual (Litron Laboratories, Rochester, New York).

Evaluation criteria:

Evaluation Criteria:
A test was considered valid if all of the following conditions were met:
• The range of MN-RET values in the negative controls were within reasonable limits of the recent laboratory background range.
• There was a significant increase in the incidence of MN-RET in the positive control treatment as compared to the concurrent negative controls.
• The mean for percent RET value in one or more of the test material treated groups was ≥ 20% of the control value indicating no undue effect on erythropoiesis (toxicity).

A test material was considered positive in this assay if the following criterion was met:
• Statistically significant increase in MN-RET frequency at one or more dose levels accompanied by a dose response.
• The statistically significant increase is out of historical range.

A test material was considered negative in this assay if the following criterion was met:
• No statistically significant dose-related increase in MN-RET when compared to the negative control.

A test result not meeting the criteria for either the positive or the negative response was considered to be equivocal.

Statistics:

MN-RET and percent RET was tested for equality of variance using Bartlett's test (alpha = 0.01; Winer, 1971). The raw data and the following transformations were examined; the common log, the inverse, and the square root in that order. The results from Bartlett's test were significant for MN-RET and percent RET in the positive control versus negative control data set and the data were log transformed to obtain equality of the variances.
The MN-RET data and the data on percent RET were analyzed by a one-way analysis of variance (Winer, 1971). The dose effect was not significant for the negative control versus treated groups analysis so Dunnett’s t-test was not performed nor were, (Winer 1971) linear dose-related trend tests performed. The alpha level at which all tests were conducted was 0.05.
The MN-NCE was not analyzed statistically and was only used as an adjunct end point to evaluate the biological significance of the MN-RET results.
The final interpretation of biological significance of the responses was based on both statistical outcome and scientific judgment.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

All dose levels tested.

Toxicity:

yes

Remarks:

Animals dosed at 0, 375 or 750 mg/kg bw/day had no indications of toxicity during the in-life portion of the MNT. One mouse dosed with 1500 mg/kg bw/day had urine perineal soiling on day two of dosing which resolved by day three.

Vehicle controls validity:

valid

Negative controls validity:

other: Vehcle control used as the negative control.

Positive controls validity:

valid

Remarks on result:

other: Results for Micronucleus Assay (MNT)

Additional information on results:

Dose Range-Finding Test (RFT):
Phase I:
Targeted dose levels of 1000 and 2000 mg/kg bw/day 2-butoxyethyl benzoate were used in the initial phase of the RFT using male and female mice. None of the mice (male and female) dosed with 1000 mg/kg bw/day displayed clinical observations, or significant changes in body weight or body temperature. One female mouse dosed with 1000 mg/kg bw/day displayed a slight decrease in body temperature of 2.2°C on day one approximately two hours after receiving the first dose.
Two of the male mice dosed with 2000 mg/kg bw/day displayed no clinical observations but had decreases in body temperature. One male mouse had a body temperature decrease of 3.6 ºC two hours after the first dose, but had mostly recovered by day two of dosing. The second male mouse had a body temperature decrease of 4.4 ºC two hours after the first dose, and lost 2.0 g of body weight over the course of the study but survived until the end of the study with no clinical observations or further substantial decreases in body temperature. The third male mouse dosed with 2000 mg/kg bw/day displayed red-colored urine on day two of dosing, but had fully recovered by day three with no further clinical signs or substantial decreases in body temperature.
The first female mouse dosed with 2000 mg/kg bw/day displayed no clinical observations or significant changes in body weight or body temperature. The next female mouse displayed no clinical observations or changes in body weight, but had a slight decrease in body temperature of 2.6°C two hours and five hours after receiving the first dose. The third female mouse dosed with 2000 mg/kg bw/day displayed red urine, dehydrated skin, slow and labored respiration, decreased feces, was absent of behavior, and had a body temperature decrease of 9.0 ºC approximately two hours after receiving the second dose at which point, the animal was declared moribund and euthanized. Gross necropsy observations by a pathologist revealed the lungs, adrenals, and urinary bladder as diffuse and red; red urine soiling was also observed. There was no evidence of gavage error.

Phase II:
Based on the mortality and clinical observations observed in the mice dosed with 2000 mg/kg bw/day, a lower dose level of 1750 mg/kg bw/day was given to additional mice (three/sex/dose). The first male mouse had decreased feces on day 3, but had no decreases in body weight or body temperature. The second male mouse had a body temperature decrease of 4.4 ºC and exhibited red urine approximately two hours after receiving the first dose. On day two, approximately five hours after the second dose, the same animal displayed red urine, dehydrated skin, slow respiration, had a body temperature decrease of 14.2 ºC, and was absent of behavior. The animal was declared moribund, was euthanized and examined by a pathologist where the bladder was observed as being diffuse and red in color, the spleen was enlarged and dark, and red urine perineal soiling was also observed. It was determined that no gavage error had occurred. The third male mouse dosed with 1750 mg/kg bw/day displayed red urine and had a body temperature decrease of 3.3 ºC on day one of dosing, but had mostly recovered by day two. On day three decreased feces were observed, but the animal had largely recovered by day four with no further clinical observations or decreases in body weight or body temperature.
The first female mouse dosed with 1750 mg/kg/bw/day had no significant changes in body weight, but had a body temperature decrease of 5.5 ºC approximately five hours after receiving the first dose, but had fully recovered by day two. This same animal was observed as having decreased feces on day three, which persisted through to day four. The next female mouse dosed with 1750 mg/kg/bw/day had a body temperature decrease of 4.3 ºC approximately two hours after receiving the first dose, and after five hours, the temperature had not recovered and was down 3.4 ºC. This same animal was observed with red urine on day one, which had resolved by day two and decreased feces on day three, which had resolved by day four. The last female animal dosed with 1750 mg/kg/bw/day had a body temperature decrease of 4.2 ºC approximately five hours after the second dose, this same animal was also observed has having decreased feces on day three, which persisted through to day four.

Phase III:
Based on the mortality and clinical observations observed in the mice dosed with 1750 mg/kg/bw/day, a lower dose of 1500 mg/kg/bw/day was given to additional mice (three/sex/dose).
All male mice dosed with 1500 mg/kg/bw/day had no significant changes in body weight or body temperature, but displayed decreased feces periodically throughout the study. All female mice dosed with 1500 mg/kg/bw/day had no significant changes in body weight or body temperature. The first female had decreased feces on day three, which had resolved by day 4. The second female mouse had red urine on day one, which was resolved by day two. On day three, the same animal had decreased feces, which had resolved by day four. The last female mouse dosed with 1500 mg/kg/bw/day, was observed with red urine on day one, which had resolved by day two with no further clinical observations.

Micronucleus Assay (MNT):

Based upon the results of the range-finding study where no appreciable differences were noted between sexes, only males were evaluated in the main study and the maximum tolerated dose was determined to be a dose of 1500 mg/kg bw/day. The middle- and low doses were 750 mg/kg bw/day and 375 mg/kg bw/day, respectively. The analytically determined concentrations of the test material in the dosing solutions used for the micronucleus test ranged from 91.5% to 102.2%% of the targeted values.

The treatments did not have a remarkable effect on the body weight or body temperature of the animals. Animals dosed at 0 (negative control), 375 mg/kg bw/day, or 750 mg/kg bw/day had no indications of toxicity based upon daily observations during the in-life portion of the MNT. One mouse dosed with 1500 mg/kg bw/day, demonstrated urine perineal soiling on day two of dosing, which was resolved by day three, with no further clinical observations. No other mice dosed at 1500 mg/kg bw/day displayed any clinical signs of toxicity throughout the study.

There were no significant differences in MN-RET frequencies between the groups treated with the test material and the negative control. The adequacy of the experimental conditions for the detection of induced micronuclei was ascertained from the observation of a significant increase in the frequencies of micronucleated RET in the positive control group.

The percent RET values observed in the test material-treated animals were not significantly different from the negative control values. The percent RET values of the positive control animals were found to be significantly lower than those of the negative control animals.

Conclusions:

2-butoxyethyl benzoate administered orally with two consecutive daily doses of ≥1500 mg/kg bw/day resulted in lethality and clear indications of systemic toxicity including an increase in the percent reticulocytes, indicating systemic availability of the test material. These data generated in the range-finding portion of the study indicate that 1500 mg/kg bw/day was the maximum tolerated dose (MTD) under the conditions of this study. Data from the RFT clearly indicated systemic availability of the test material when administered by oral gavage. Based upon the results of the study reported herein, it was concluded that the 2-butoxyethyl benzoate, did not induce a significant increase in the frequency of micronucleated reticulocytes in the peripheral blood when given as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. Hence, 2-butoxyethyl benzoate is considered negative in this test system under the experimental conditions used.

Executive summary:

The in vivo genotoxic potential of 2-butoxyethyl benzoate was evaluated by examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood. The test material was administered to male Crl:CD1(ICR) mice by single oral gavage on two consecutive days at dose levels of 0 (negative control), 375, 750, and 1500 mg/kg body weight (bw). The highest dose level was based upon the results of a range-finding test where at higher doses treatment-related deaths were observed in male and female mice.

The analytically determined concentrations of the test material in the dosing solutions used for the first day of dosing in the micronucleus ranged from 91.5% to 102.2% of the targeted concentrations. All animals were observed for clinical signs prior to dosing and at 2, 5, and 24 hours following each dosing. Groups of animals were euthanized 48 hours after the second treatment for the collection of peripheral blood and evaluation of RET (approximately 5,000/animal) for MN by flow cytometry. The proportion of RET was also determined based upon 5,000 RET per animal and the results expressed as a percentage. Mice treated with 40 mg/kg bw cyclophosphamide monohydrate by a single gavage dose and euthanized 48 hours later served as positive controls.

There were no treatment-related deaths or treatment-related clinical signs in the observation period of the definitive micronucleus test. There were no statistically significant increases in the frequencies of MN-RET or statistically significant effects on the percent RET in groups treated with the test material as compared to the negative controls. There was a significant increase in the frequency of MN-RET and a decrease in the percentage of RET in the positive control chemical group as compared to the negative control group. Based upon the results of the study reported herein, 2-butoxyethyl benzoate is considered negative in this test system under the experimental conditions used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic Toxicity In Vitro:

OECD Guideline 471 (Bacterial Reverse Mutation Assay):

In a confirmatory mutation assay, 2 -butoxyethyl benzoate was exposed in triplicate at 32, 101, 320, 1013 and 3200 µg/plate test doses with TA98, TA100, TA1535 and TA1537 and at 50, 158, 500, 1580 and 5000 µg/plate test doses with WP2uvrA (pKM101) along with the vehicle and appropriate positive controls. The test substance did not precipitate on the basal agar plates at any of the tested doses. There was no positive mutagenic response observed in any of the strains in any of the tested doses either in the presence or in the absence of metabolic activation. Under the conditions of the current study, the test substance, 2 -butoxyethyl benzoate was negative (nonmutagenic) in thisSalmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay.

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

2-Butoxyethyl benzoate was evaluated in the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 26.6 to 425 μg/ml in the absence of S9 and in the presence of S9. The highest concentration was based on limit of solubility of the test material in the treatment medium. There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the vehicle control cultures in either the absence or presence of S9. The results of the CHO/HGPRT forward gene mutation assay with 2-Butoxyethyl benzoate indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test).

2-Butoxyethyl benzoate was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 425.0 μg 2-Butoxyethyl benzoate per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The duration of treatment was 4 or 24 hours without S9 and 4 hours with S9. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon solubility of the test material and the mitotic index values. In this study cultures treated for 4 hours with targeted concentrations of 0 (vehicle control), 26.6, 106.3, and 425.0 μg/ml in the presence of S9, 0 (vehicle control), 90.0, 130.0, and 190.0 μg/ml in the absence of S9, and cultures treated for 24 hours with 0 (vehicle control), 90.0, 130.0 and 170.0 μg/ml in the absence of S9 were analyzed for chromosomal aberrations.

There were no significant increases in the frequency of cells with aberrations administered 2-Butoxyethyl benzoate in either the absence or presence of S9 activation. Based upon these results, 2-Butoxyethyl benzoate was considered to be negative in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Genetic Toxicity In Vivo:

OECD Guideline 474 (Mammailan Erythrocyte Micronucleus Test):

The in vivo genotoxic potential of 2-butoxyethyl benzoate was evaluated by examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood. The test material was administered to male Crl:CD1(ICR) mice by single oral gavage on two consecutive days at dose levels of 0 (negative control), 375, 750, and 1500 mg/kg body weight (bw). The highest dose level was based upon the results of a range-finding test where at higher doses treatment-related deaths were observed in male and female mice.

The analytically determined concentrations of the test material in the dosing solutions used for the first day of dosing in the micronucleus ranged from 91.5% to 102.2% of the targeted concentrations. All animals were observed for clinical signs prior to dosing and at 2, 5, and 24 hours following each dosing. Groups of animals were euthanized 48 hours after the second treatment for the collection of peripheral blood and evaluation of RET (approximately 5,000/animal) for MN by flow cytometry. The proportion of RET was also determined based upon 5,000 RET per animal and the results expressed as a percentage. Mice treated with 40 mg/kg bw cyclophosphamide monohydrate by a single gavage dose and euthanized 48 hours later served as positive controls.

There were no treatment-related deaths or treatment-related clinical signs in the observation period of the definitive micronucleus test. There were no statistically significant increases in the frequencies of MN-RET or statistically significant effects on the percent RET in groups treated with the test material as compared to the negative controls. There was a significant increase in the frequency of MN-RET and a decrease in the percentage of RET in the positive control chemical group as compared to the negative control group. Based upon the results of the study reported herein, 2-butoxyethyl benzoate is considered negative in this test system under the experimental conditions used.

Justification for classification or non-classification

Based upon the negative results in both the In Vitro and In Vivo key studies, classification according to GHS is not warranted for 2 -butoxyethyl benzoate.