N-butyl phenyl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity and Mammalian cell clastogenicity

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 29, 2016 to October 22, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test Item Name: Butyl Phenyl Ether
Appearance/Colour/Odour: Colourless liquid
Chemical Name: Butoxy Benzene
CAS Number: 1126-79-0
Molecular Weight: 150.2
Lot N°: 06027KH
Analysed Purity: (Provided by Sponsor) 99.5 %
Supplied to JRF by: The Dow Chemical Company, Midland, Michigan
Manufactured by: Not provided
Date of Manufacture: January 27, 2016
Re-Certification Date: January 19, 2017
Storage Condition (at JRF): As per the instruction received from the Sponsor on storage of the test item, the test item was stored at:
Storage Temperature : Ambient Temperature
Storage Container : In original container as supplied by the sponsor
Storage Location : Test Item Control Office, JRF

Target gene:

This assay measures the ability of the test item to induce reverse mutations at specific histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100, and at tryptophan locus in Escherichia coli WP2uvrA (pKM101), which are known for their reliability and reproducibility in a short term mutagenicity assay and are also recommended by the OECD, EPA and other guidelines.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Based upon the inhibition observed in the Initial Toxicity Mutation Assay, in the Confirmatory Mutation Assay, bacterial cultures were exposed to Butyl Phenyl Ether at concentrations of 2.3, 4.7, 9.4, 18.8, 37.5, 75, and 150 μg/plate in the absence of metabolic activation system and 9.4, 18.8, 37.5, 75, 150, 300, and 600 μg/plate in the presence of metabolic activation system (10% v/v S9 mix) for all tester strains (three plates/concentration).

Vehicle / solvent:

Butyl Phenyl Ether was tested in the absence and presence of metabolic activation using dimethyl sulfoxide (DMSO) as the solvent. Butyl phenyl ether was found to be stable in DMSO at room temperature for at least 4 hours. Butyl Phenyl Ether was found to be insoluble in sterile distilled water, while it was found to be soluble in DMSO at a concentration of 50000 μg/mL.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 9-Aminoacridine hydrochloride hydrate (CAS N° 52417-22-8); 2-Aminoanthracene (CAS N° 613-13-8)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation assay

Number of Replicates:
There will be two replicates for the initial toxicity-mutation assay and three replicates for the confirmatory mutation assay.

Plating Procedure:
The initial toxicity-mutation, as well as the confirmatory mutation assay will be conducted using the preincubation assay method.
A. Presence of metabolic activation
a) 50 or 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) 50 or 100 μL test dose/vehicle/appropriate positive control
b) 100 μL bacterial culture
c) 500 μL of 0.2 M phosphate buffer

These test constituents will be transferred into sterile test tubes and will be kept in an incubator shaker/shaking water bath for approximately 20 ± 2 minutes at 37 ± 1 ºC. After this period, 2 mL of soft agar containing histidine-biotin / tryptophan will be added to each of the tubes. These constituents will be overlaid onto VB agar plates. After the soft agar sets, the plates will be incubated at 37 ± 1°C for 48 to 72 hours.

Justification for Selection of the Test System:
This assay measures the ability of the test item to induce reverse mutations at specific histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100, and at tryptophan locus in Escherichia coli WP2uvrA (pKM101), which are known for their reliability and reproducibility in a short term mutagenicity assay and are also recommended by the OECD, EPA and other guidelines.

Rationale for test conditions:

In the Initial Toxicity-Mutation Assay, bacterial cultures were exposed to Butyl Phenyl Ether at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (two plates/concentration). Precipitation was observed at the concentration of 5000 μg/plate, which did not interfere with the colony counting in the absence and presence of metabolic activation in all of the five strains. Complete inhibition (with 100% reduction in revertant colonies) was observed at the tested concentrations of 150 – 5000 μg/plate in the absence of metabolic activation system and 500 – 5000 μg/plate in the presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. Partial inhibition with reduction in number of revertant colonies (34- 46%) was observed at the tested concentration of 50 μg/plate in absence of metabolic activation system in all the tester strains. Normal growth was observed in all tester strains at the tested concentrations of 1.5 – 15 μg/plate in the absence of the metabolic activation system and 1.5 – 150 μg/plate in the presence of the metabolic activation system. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.
From the results of the Initial Toxicity-Mutation Assay, 150 μg/plate was selected in absence of metabolic activation system and 600 μg/plate was selected in the presence of metabolic activation system for all tester strains, being the highest concentration for the Confirmatory Mutation Assay.

Evaluation criteria:

Assay Evaluation Criteria:
Once criteria for a valid assay have been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were that 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 article either in the absence or presence of the metabolic activation system.

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 negative control value.

Strains TA100 and Escherichia coli WP2uvrA (pKM101):
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0- times the mean negative control value.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was determined to be non mutagenic.

Statistics:

The following statistical treatments were used in this study: Means, standard deviations and relative standard deviations.

Key result

Species / strain:

other: Salmonella typhimurium strains TA1537, TA1535, TA98, and TA100 and Escherichia coli WP2uvrA (pKM101)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All doses in all tester strains.

Cytotoxicity / choice of top concentrations:

other: Compete inhibition of background lawn at 150 μg/plate and partial inhibition of background lawn with reduction in revertant colonies (42-54%) at 75 μg/plate in all tester strains. All other doses were normal in all tester strains.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: Salmonella typhimurium strains TA1537, TA1535, TA98, and TA100 and Escherichia coli WP2uvrA (pKM101)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

All doses in all tester strains.

Cytotoxicity / choice of top concentrations:

other: Complete inhibition of background lawn at 600 μg/plate and partial inhibition of background lawn with reduction in revertant colonies (45-52%) at 300 μg/plate in all tester strains. All other doses were normal in all tester strains.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Negative Controls:
The results of the study indicate that the values for the negative controls (DMSO) in all strains were within limits of historical range.

Positive Controls:
2-Aminoanthracene was used as the positive control in the presence of a metabolic activation for all the tester strains during the Initial Toxicity Mutation Assay and Confirmatory Mutation Assay. Historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation. Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were within the historical ranges. This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay. An increase in the mean number of revertants was not observed in Salmonella typhimurium tester strain TA100 (Initial Toxicity Mutation Assay and Confirmatory Mutation Assay) treated with 2-aminoanthracene in the absence of metabolic activation, but a clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Initial Toxicity Mutation Assay:
Bacterial cultures were exposed to Butyl Phenyl Ether at concentrations of 1.5, 5, 15, 50, 150, 500, 1500, and 5000 μg/plate (two plates/concentration) in the absence and presence of the metabolic activation system (5% v/v S9 mix). Precipitation was observed at the concentration of 5000 μg/plate, which did not interfere with the colony counting in the absence and presence of metabolic activation in all of the five strains. Complete inhibition of background lawn (with 100% reduction in revertant colonies) was observed at the tested concentrations of 150 – 5000 μg/plate in the absence of metabolic activation system and 500 – 5000 μg/plate in the presence of the metabolic activation system in all the tester strains. Partial inhibition of background lawn with reduction in number of revertant colonies (34- 46%) was observed at the tested concentration of 50 μg/plate in absence of metabolic activation system in all the tester strains. Normal growth was observed in all tester strains at the tested concentrations of 1.5 – 15 μg/plate in the absence of the metabolic activation system and 1.5 – 150 μg/plate in the presence of the metabolic activation system. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

Confirmatory Mutation Assay:
From the results of the Initial Toxicity-Mutation Assay, 150 μg/plate was selected in the absence of metabolic activation system and 600 μg/plate was selected in the presence of metabolic activation system for all tester strains, being the highest concentrations for the confirmatory mutation assay. In the Confirmatory Mutation Assay, bacterial cultures were exposed to Butyl Phenyl Ether at concentrations of 2.3, 4.7, 9.4, 18.8, 37.5, 75 and 150 μg/plate in the absence of metabolic activation system and 9.4, 18.8, 37.5, 75, 150, 300, and 600 μg/plate in the presence of metabolic activation system (10% v/v S9 mix) for all tester strains (three plates/concentration). Complete inhibition of background lawn (with 100% reduction in revertant colonies) was observed at the tested concentration of 150 μg/plate in the absence of metabolic activation system and at the tested concentration of 600 μg/plate in the presence of metabolic activation system in all tester strains.
Partial inhibition was observed at the tested concentration of 75 μg/plate in the absence of metabolic activation system and at the tested concentration of 300 μg/plate in the presence of metabolic activation system in all tester strains. Reduction in number of revertant colonies in tester strains TA1537 (42% and 52%), TA1535 (53% and 51%), TA98 (54% and 50%), TA100 (45% and 47%) and Escherichia coli WP2uvrA (pKM101) (47% and 45%) was observed at the tested concentration of 75 μg/plate in the absence of metabolic activation and at the tested concentration of 300 μg/plate in the presence of metabolic activation, respectively. Normal growth was observed at other tested concentrations in the absence and presence of the metabolic activation system in all tester strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

Dose Formulation Analysis:

The Butyl Phenyl Ether stock concentrations 23, 47, 94, 188, 375, 750, 1500, 3000 and 6000 μg/mL was found to be within acceptable range of ± 15% of nominal (% RSD < 10%) during confirmatory mutation assay. The 0 hour concentrations of butyl phenyl ether in the dose formulation were found to be 98.4%, 92.1%, 97.0%, 91.0%, 94.4%, 95.0%, 92.8%, 94.3% and 90.5% of dose level 23 μg/mL, 47 μg/mL, 94 μg/mL, 188 μg/mL, 375 μg/mL, 750 μg/mL, 1500 μg/mL, 3000 μg/mL and 6000 μg/mL, respectively. The concentrations of butyl phenyl ether in the dose formulation after 4 hours were found to be 105% and 106% of the 0 hour concentrations of dose level T1 (23 μg/mL) and T7 (6000 μg/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

Conclusions:

From the results of this study, under the specified experimental conditions, Butyl Phenyl Ether is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101).

Executive summary:

The potential of Butyl Phenyl Ether to induce reverse mutations in Salmonella typhimurium strains TA1537, TA1535, TA98, and TA100 and a tryptophan deficient strain, Escherichia coli WP2uvrA(pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method.

Butyl Phenyl Ether was tested in the absence and presence of metabolic activation using dimethyl sulfoxide (DMSO) as the solvent. In the Initial Toxicity-Mutation Assay, bacterial cultures were exposed to Butyl Phenyl Ether at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (two plates/concentration). Precipitation was observed at the concentration of 5000 μg/plate, which did not interfere with the colony counting in the absence and presence of metabolic activation in all of the five strains. Complete inhibition (with 100% reduction in revertant colonies) was observed at the tested concentrations of 150 – 5000 μg/plate in the absence of metabolic activation system and 500 – 5000 μg/plate in the presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. Partial inhibition with reduction in number of revertant colonies (34- 46%) was observed at the tested concentration of 50 μg/plate in absence of metabolic activation system in all the tester strains. Normal growth was observed in all tester strains at the tested concentrations of 1.5 – 15 μg/plate in the absence of the metabolic activation system and 1.5 – 150 μg/plate in the presence of the metabolic activation system.

No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

From the results of the Initial Toxicity-Mutation Assay, 150 μg/plate was selected in absence of metabolic activation system and 600 μg/plate was selected in the presence of metabolic activation system for all tester strains, being the highest concentration for the Confirmatory Mutation Assay.

In the Confirmatory Mutation Assay, bacterial cultures were exposed to Butyl Phenyl Ether at concentrations of 2.3, 4.7, 9.4, 18.8, 37.5, 75, and 150 μg/plate in the absence of metabolic activation system and 9.4, 18.8, 37.5, 75, 150, 300, and 600 μg/plate in the presence of metabolic activation system (10% v/v S9 mix) for all tester strains (three plates/concentration). Complete inhibition of background lawn (with 100% reduction in revertant colonies) was observed at the tested concentration of 150 μg/plate in the absence of metabolic activation system and at the tested concentration of 600 μg/plate in the presence of metabolic activation system in all the tester strains. Partial inhibition of background lawn with reduction in revertant colonies (42-54%) was observed at the tested concentration of 75 μg/plate in the absence of metabolic activation system in all tester strains. Partial inhibition of background lawn with reduction in revertant colonies (45-52%) was observed at 300 μg/plate in presence of metabolic activation system in all tester strains. Normal growth was observed at other tested concentrations in the absence and presence of metabolic activation system in all tester strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when

compared with the concurrent negative control.

All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

The Butyl Phenyl Ether stock concentrations 23, 47, 94, 188, 375, 750, 1500, 3000 and 6000 μg/mL was found to be within acceptable range of ± 15% of nominal concentrations during the Confirmatory Mutation Assay. The 0 hour concentrations of butyl phenyl ether in the dose formulation were found to be 98.4%, 92.1%, 97.0%, 91.0%, 94.4%, 95.0%, 92.8%, 94.3% and 90.5% of dose level 23 μg/mL, 47 μg/mL, 94 μg/mL, 188 μg/mL, 375 μg/mL, 750 μg/mL, 1500 μg/mL, 3000 μg/mL and 6000 μg/mL, respectively. The concentrations of butyl phenyl ether in the dose formulation after 4 hours were found to be 105% and 106% of the 0 hour concentrations of dose level T1 (23 μg/mL) and T7 (6000 μg/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, Butyl Phenyl Ether is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2uvrA (pKM101).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 4, 2016 to August 5, 2016

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:

EPA OPPTS 870.5375 - 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

Type of assay:

other: In-vitro determination of chromosomal aberration frequency and incidence of polyploidy

Specific details on test material used for the study:

Test Material Name: Butyl phenyl ether
Chemical Name: Butoxybenzene
Synonyms: None
Lot/Reference/Batch Number: 06027KH
Purity/Characterization (Method of Analysis and Reference): The non-GLP certificate of analysis lists the purity as 99.5% by gas liquid chromatography with structure confirmation by infrared spectroscopy (Rajzer, 2007).
Stability: Butyl phenyl ether, lot 22466, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under U.S. EPA OPPTS Guideline 830.6317 (Ferruzzi, 2016).

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: 10-12 weeks

Lymphocyte Cultures:
The animals were euthanized with carbon dioxide just prior to collecting the samples by cardiac puncture. Blood, treated with an anticoagulant (e.g., heparin), from several rats was pooled for sample collection. Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heat-inactivated fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone 0.25 μg/ml), and an additional 2 mM L-glutamine) in addition with 30 μg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C. Treatment medium was the above-mentioned medium without serum and was used during the 4-hour treatment conditions, while complete medium was used for the 24-hour treatment condition.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg body weight) male Sprague Dawley 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 1502.0 μg butyl phenyl ether per ml of culture medium. The highest concentration was based on the limit concentration specified in the guideline (10 mM).

Vehicle / solvent:

Dimethyl sulfoxide (DMSO, CAS No. 67-68-5) was selected as the solvent used to dissolve the test material and was used as the vehicle control.

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: Cyclophosphamide monohydrate

Details on test system and experimental conditions:

Lymphocyte Cultures:
The animals were euthanized with carbon dioxide just prior to collecting the samples by cardiac puncture. Blood, treated with an anticoagulant (e.g., heparin), from several rats was pooled for sample collection. Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heat-inactivated fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone 0.25 μg/ml), and an additional 2 mM L-glutamine) in addition with 30 μg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C. Treatment medium was the above-mentioned medium without serum and was used during the 4-hour treatment conditions, while complete medium was used for the 24-hour treatment condition.

In Vitro Metabolic Activation System:
S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg body weight) male Sprague Dawley rats were purchased from a commercial source 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, 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 treatment 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 medium was 1/5 of the concentrations stated above.

Preparation of the Treatment Solution and Administration of the Test Material:
The test material was found to be soluble in DMSO up to 150.4 mg/ml. 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 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 treatment medium, 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 gas chromatography with mass spectrometry detection (GC/MS). Analytical method validation was performed concurrently with sample analysis. 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 and positive controls should be within the control limits of the laboratory historical control values as calculated using previous laboratory values. A test chemical was considered positive in this assay if it induced a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was outside the control limits of the laboratory historical vehicle control range. A test chemical was considered negative in this assay if it did not induce a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was not outside the control limits of the laboratory historical vehicle control range. If a test chemical did not meet either of the above criteria it may have been considered equivocal.

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, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at alpha = 0.05, 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 were 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:

positive

Remarks:

Statistical analysis of the data identified a significant difference between the aberration frequency of the vehicle control and the 55.6 μg/ml treatment concentration with a positive linear trend (alpha = 0.05).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

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 (50.0, 70.0 and 90.0 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).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Rat

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Statistical analysis of the data did identify a significant difference between the vehicle control and the highest two concentrations (i.e., 50.0 and 60.0 μg/ml) of the test material with a positive linear trend (alpha = 0.05).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

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 1504 μg/ml of the test material and medium containing 1% DMSO was 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 treatment medium with solvent alone (treatment medium with the test material, pH = 7.39, osmolality = 446 mOsm/kg H2O; treatment medium with 1% DMSO, pH = 7.48, osmolality = 434 mOsm/kg H2O).

Remarks on result:

other: Assay A1 4-Hour Treatment

Assay A1:

Cultures were treated with the test material for 4 hours in the absence and presence of S9 activation at concentrations of 0 (vehicle control), 2.1, 6.2, 18.5, 55.6, 166.9, 500.7, and 1502.0 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 0.7 μg/ml. The test material precipitated in the treatment medium at the top three concentrations (i.e., 166.9, 500.7, and 1502.0 μg/ml) in all treatment conditions, as observed at the end of treatment. Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 88.7 to 136.0% of the target and verified that concentrations used for treatment were within the acceptable range.

Short Treatment:

In the absence of S9, the cultures displayed excessive toxicity in the three highest treatment concentrations (i.e., 166.9, 500.7, and 1502.0 μg/ml) as indicted by the lack of observable mitotic figures. The remaining cultures had relative mitotic indices ranging from 87.4 to 67.9% compared to the vehicle control values. Based upon these results, this portion of the assay was repeated in Assay B1, as a result of not achieving the recommended level of cytotoxicity (i.e. 60 + 10% reduction in mitotic index).

In the presence of S9, excessive toxicity was also observed in the three highest treatment concentrations (i.e., 166.9, 500.7, and 1502.0 μg/ml). The remaining cultures had relative mitotic indices ranging from 92.2 to 30.4% as compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0.0 (vehicle control), 2.1, 18.5, and 55.6 μg/ml were selected for the determination of chromosomal aberration frequency and incidence of polyploidy in the presence of S9 activation.

Among the cultures treated with the positive control chemicals for 4 hours, 2 μg/ml of CP was selected for evaluation of aberrations in the presence of S9.

In the presence of S9, there were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

In the assay with S9, cultures treated with the test material at concentrations of 2.1, 18.5, and 55.6 μg/ml had aberrant cell frequencies of 1.0, 3.3, and 7.7%, respectively as compared to the vehicle control value of 1.3%. Statistical analysis of these data identified a significant difference between the aberration frequency of the vehicle control and the 55.6 μg/ml treatment concentration with a positive linear trend (alpha = 0.05). The aberration frequency observed in the cultures treated with 55.6 μg/ml of the test material was outside of the laboratory

historical vehicle control range. The frequencies of aberrant cells observed in the remaining test material treated cultures were within the laboratory historical control range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical. Aberrant cell frequencies in CP (2.0 μg/ml, +S9) treatment cultures was 38.7%. The positive control value was within the control limits of the laboratory historical positive control range.

Continuous Treatment:

Cultures treated continuously for 24 hours in the absence of S9 activation had excessive toxicity in the three highest treatment concentrations (i.e., 166.9, 500.7, and 1502.0 μg/ml) as i ndicated by the lack of observable mitotic figures. The remaining cultures had relative mitotic indices ranging from 62.9 to 94.8% relative to the vehicle control value. The guideline recommended level ofcytotoxicity (i.e. 60 + 10% reduction in mitotic index) was not achieved in the 24 hour treatment condition in the absence of S9.

Assay B1:

Cultures were treated with the test material in the absence of S9 for 4 hours at target concentrations of 0.0 (vehicle control), 2.0, 20.0, 50.0, 70.0, 90.0, 110.0, and 160.0 μg/ml. The test material precipitated in the treatment medium at the top three concentrations (i.e., 90.0, 110.0, and 160.0 μg/ml) as observed at the end of treatment. Cultures were treated with the test material in the presence of S9 for 4 hours at target concentrations of 0.0 (vehicle control), 2.0, 20.0, 30.0, 40.0, 50.0, 60.0, and 160.0 μg/ml.

Short Treatment:

In the absence of S9, the cultures displayed excessive toxicity in the two highest concentrations (i.e., 110.0 and 160.0 μg/ml) as indicated by the lack of observable mitotic figures. The remaining cultures had relative mitotic indices ranging from 41.5 to 130.5% compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0.0 (vehicle control), 50.0, 70.0, and 90.0 μg/ml were selected for the determination of chromosomal aberration frequency and incidence of polyploidy in the absence of S9 activation.

In the presence of S9, excessive toxicity was also observed the highest treatment concentration (i.e., 160.0 μg/ml). The remaining cultures had relative mitotic indices ranging from 40.5 to 100.0% as compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 2.0, 50.0, and 60.0 μg/ml were selected for the determination of chromosomal aberration frequency and incidence of polyploidy in the presence of S9 activation.

Among the cultures treated with the positive control chemicals, 0.5 μg/ml MMC (-S9,) and 2 μg/ml of CP (+S9,) were selected for evaluation of aberrations.

There were no significant increases in the incidence of polyploid cells in the test material treated cultures as compared to the vehicle control.

In the non-activation assay, the frequency of cells with aberrations in the vehicle control was 0.7% and the corresponding values at target concentrations of 50.0, 70.0, and 90.0 μg/ml were 0.3, 0.7, and 0.3%, respectively. Statistical analysis of these data did not identify any significant differences between the vehicle control and any of the test material treated cultures. The frequencies of cells with aberrations in all cultures were within laboratory historical vehicle control values.

In the presence of S9, the frequency of cells with aberrations in the vehicle control was 0.7% and the corresponding values at target concentrations of 2.0, 50.0, and 60.0 μg/ml were 0.0, 4.7, and 7.0, respectively. Statistical analysis of these data did identify a significant difference between the vehicle control and the highest two concentrations (i.e., 50.0, and 60.0 μg/ml) of the test material with a positive linear trend (alpha = 0.05). The frequencies of aberrations observed in the two highest target concentrations were outside the historical vehicle control range. The frequency of aberrations in the remaining target concentration (i.e., 2.0 μg/ml) was within the historical vehicle control range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in MMC (0.5 μg/ml, -S9) and CP (2.0 μg/ml, +S9) treated cultures were 24.7% and 15.7%, respectively. The aberration frequencies observed in the positive control treated cultures were within the laboratory historical positive control range.

Continuous Treatment:

Due to the positive findings in the 4-hour treatment condition the 24-hour treatment portion of the assay was not evaluated.

Conclusions:

It was concluded that under the experimental conditions used, butyl phenyl ether was considered clastogenic in this in vitro chromosomal aberration assay utilizing rat lymphocytes as a result of the clear positive response observed in the 4 hour treatment (with S9).

Executive summary:

Butyl phenyl ether (Butoxybenzene) 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 1502.0 μg butyl phenyl ether per ml of culture medium. The highest concentration was based on the limit concentration specified in the guideline (10 mM). The duration of treatment was 4 hours without and with S9 and 24 hours without S9. The analytically determined concentrations of butyl phenyl ether in the dose preparations ranged from 88.7 to 136.0% of the targeted values. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon cytotoxicity of the test material. In the initial cytogenetic assay cultures treated for 4 hours with targeted concentrations of 0.0 (vehicle control), 2.1, 18.5, and 55.6 μg/ml in the presence of S9 were selected for determining the incidence of chromosome aberrations based upon cytotoxicity. At the highest dose (55.6 μg/ml) a statistically significant increase in the frequency of aberrant cells was observed (1.3% in the vehicle control vs. 7.7% at 55.6 μg/ml), and the observed increase was dose-dependent. This frequency of aberrant cells at 55.6 μg/ml was outside of the laboratory’s historical control range. In the initial assay the 4 and 24 hour treatment conditions in the absence of S9 the guideline required level of cytotoxicity (60 + 10%) was not achieved, therefore, these cultures were not evaluated for chromosome aberration frequency.

In the repeat assay cultures were treated with concentrations ranging from 0.0 (vehicle control) to 160 μg/ml for 4 hours, in the absence and presence of S9. Based upon the mitotic indices, cultures treated in the absence of S9, with targeted concentrations of 0 (vehicle control), 50.0, 70.0, and 90.0 μg/ml and cultures treated in the presence of S9, with targeted concentrations of 0.0 (vehicle control), 2.0, 50.0, and 60.0 μg/ml were selected for determining the incidence of chromosomal aberrations.

There were no statistically significant increases in the frequencies of cells with aberrations in the cultures treated for 4 hours in the absence of S9 when compared to the vehicle control cultures. There was a significant increase in the frequency of cells with aberrations in the presence of S9, with 0.7% in the vehicle control and 4.7% and 7.0% at the highest two concentrations (i.e., 50.0 and 60.0 μg/ml), respectively. The 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. The vehicle and positive controls in all assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay. As a result of the clear positive response observed in the 4 hour treatment (with S9) the 24 hour treatment condition was not evaluated.

Based upon these results, butyl phenyl ether was considered to be positive for clastogenicity in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo rat bone marrow micronucleus assay

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

Assay incorporated into OECD 422 assay

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, North Carolina)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: P - 8 weeks
- Weight at study initiation: (P) Males: 277-281 g; Females: 197-200 g
- Fasting period before study: none
- Housing: Upon arrival animals were housed two or three per cage in solid bottom stainless steel cages.
The solid bottom cages contained ground corn cob bedding. Cages contained a feed crock and a press
ure activated lixit valve-type watering system.
After assignment to study, animals were housed singly in solid bottom stainless steel cages, except durin
g breeding and during the gestation and littering phases of the study. The solid bottom cages contained
ground corn cob bedding. During breeding, one male and one female were placed in stainless steel
cages with wire mesh floors that were suspended above absorbent paper in order to better visualize
copulation plugs. During gestation and littering, dams (and their litters) were housed in plastic cages
provided with irradiated ground corn cob bedding from approximately GD 0 until completion of lactation
(LD 4). Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Enrichment: Enrichment for rats was provided from the day of arrival until necropsy. The cage contai
ned nylon bones during the pre-breeding phase (males and females) and following mating (all males) or
paper nesting material during the gestation and lactation phases of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light):12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
IN-LIFE DATES: From: To: Dosing started: June 01, 2016; Male necropsy July 6-7, 2016; Female
necropsy July 13 -19, 2016. Pups euthenized on Post natal day 4.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSE: Butyl phenyl ether was administered in corn oil and CP in distilled water, such
that a dose volume of 4 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using
the most current body weight. Dose solutions were not corrected for purity. Butyl phenyl dose solutions
were prepared within the 12 day stability limit.
VEHICLE
- Supplier: Corn Oil supplied by Sigma-Aldrich, St. Louis, Missouri
- Justification for use and choice of vehicle (if other than water): Vehicle choice determined by solubility.

Duration of treatment / exposure:

For the test material: 36 days
for the positive control a single dose 24 hours prior to scheduled necropsy.

Frequency of treatment:

daily

Post exposure period:

24 hours

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

350 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, 20 mg/kg bw/day, 5 males

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

The bone marrow from the first five male rats of the vehicle control group , the butyl phenyl ether treated groups, and the five male rats in the CP group were removed at the scheduled necropsy on test day 36 from the left femur by aspiration into fetal calf serum and centrifuged at 6000-10000 rpm for five minutes. The cell pellet was resuspended in a drop of serum and a wedge film was prepared on a microscope slide. The slides were allowed to air dry and then fixed with methanol prior to staining with Acridine Orange. Only slides from the male rats were scored since no apparent difference in toxicity (e.g., relative liver weight and effects on urinary bladder) between the sexes was observed in the study and decreased food consumption and body weight and increased reticulocyte counts were observed in males only. Slides were coded, scored, and decoded upon completion to control for bias.

Evaluation criteria:

Whenever possible, a minimum of 4000 polychromatic erythrocytes (PCE) were examined from each animal and the number of MN-PCE were recorded. The proportion of PCE among total erythrocytes (PCE and normal chromatic erythrocytes (NCE)) in the bone marrow were determined by examining at least 500 erythrocytes. The percentage of PCE was expressed as PCE /PCE+NCE x 100.

Statistics:

MN-PCE and percent PCE were tested for equality of variance using Bartlett's test (alpha = 0.01; Winer, 1971). If the results from Bartlett's test were significant, then the data for the parameter might have been subjected to a transformation to obtain equality of the variances. The transformations that were examined are the common log, the inverse, and the square root in that order. The data were reviewed and an appropriate form of the data selected and subjected to the following analysis.
The MN-PCE data and the data on percent PCE were analyzed by a one-way analysis (Winer, 1971). Pairwise comparisons of treated vs. control groups were done, if the dose effect was significant, by Dunnett’s t-test, one-sided (upper) for MN-PCE and two-sided for the percent PCE (Winer 1971). Linear dose-related trend tests would be performed if any of the pairwise comparisons yielded significant differences. The alpha level at which all tests was conducted was 0.05.
The final interpretation of biological significance of the responses was based on both statistical outcome and scientific judgment. A test was considered valid if all of the following conditions were met:
• There was a significant increase in the incidence of MN-PCE in the positive control treatment as compared to the concurrent negative controls.
• The mean for percent PCE value in one or more of the test material treated groups should be  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-PCE frequency at one or more dose levels accompanied by a dose response.
A test material was considered negative in this assay if the following criterion is met:
• No statistically significant dose-related increase in MN-PCE when compared to the negative control.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

general toxicity (systemic - liver wt increase, effects in bladder)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

The %PCE observed in the groups treated with butyl phenyl ether was not significantly different from the vehicle control group. As expected, the %PCE of the positive control group was significantly lower than that of the vehicle control group. The MN-PCE frequency (/1000) in the vehicle control group was 3.30 ± 1.10 (mean ± SD). The MN-PCE frequencies in the 30, 100, and 350 mg/kg bw/day treatment groups were 3.30 ± 1.32, 3.69 ± 0.85, and 3.72 ± 1.01, respectively. There were no significant differences in MN-PCE frequency among the groups treated with butyl phenyl ether and the vehicle control group. The adequacy of the experimental conditions for the detection of induced micronuclei was confirmed with the observation of a significant increase in the frequencies of MN-PCE in the positive control group (35.65 ± 3.74). 

The treatment-related effects on liver and urinary bladder (see below) demonstrated the systematic distribution of the test material and/or its metabolites after oral gavage. The treatment-related hematologic effects (e.g., statistically significant higher mean reticulocyte count in the 350 mg/kg/day males) further demonstrated the adequate bone marrow exposure to the test material and/or its metabolites. Therefore, butyl phenyl ether was negative in this bone marrow erythrocyte MNT under the experimental conditions used. 

Table: Summary of Micronucleated Reticulocytes (MN-RET) Frequencies and Percent Reticulocytes (%RET) - Males

Exposure (mg/kg bw/day)	N^a	MN-PCE/1000^b	% PCE^c
0^d	5	3.30 ± 1.10	63.1 ± 4.0
30	5	3.30 ± 1.32	66.4 ± 6.2
100	5	3.69 ± 0.85	63.9 ± 2.0
350	5	3.72 ± 1.01	68.4 ± 9.3
Positive Control (20 mg/kg CP^e)	5	35.65 ± 3.74 *	43.8 ± 9.7 *
^aThe number of animals per dose group for the micronucleus assay. ^bMN-PCE frequency: MN-PCE per 1000 PCEs. 4000 PCEs were examined per animal except that 712 PCEs were scored for Animal 1494 in the 100 mkd group and 2100 PCEs were scored for Animal 1508 in the 350 mkd group due to smear quality. ^cPCE percentage: 500 erythrocytes were examined per animal and % PCE = PCE/(PCE+NCE) × 100. ^dAnimals were dosed with the vehicle (corn oil). ^eCP = Cyclophosphamide monohydrate * Significantly different from the negative control (alpha=0.05).

Conclusions:

There were no treatment-related effects in micronucleated polychromatic erythrocytes (MN-PCE) frequency for males at any dose. Therefore, butyl phenyl ether was negative in this bone marrow erythrocyte MNT under the experimental conditions used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Genotoxicity:

Bacterial Mutagenicity

The potential of Butyl Phenyl Ether to induce reverse mutations in Salmonella typhimurium strains TA1537, TA1535, TA98, and TA100 and a tryptophan deficient strain, Escherichia coli WP2uvrA(pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method.

Butyl Phenyl Ether was tested in the absence and presence of metabolic activation using dimethyl sulfoxide (DMSO) as the solvent. In the Initial Toxicity-Mutation Assay, bacterial cultures were exposed to Butyl Phenyl Ether at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (two plates/concentration). Precipitation was observed at the concentration of 5000 μg/plate, which did not interfere with the colony counting in the absence and presence of metabolic activation in all of the five strains. Complete inhibition (with 100% reduction in revertant colonies) was observed at the tested concentrations of 150 – 5000 μg/plate in the absence of metabolic activation system and 500 – 5000 μg/plate in the presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. Partial inhibition with reduction in number of revertant colonies (34- 46%) was observed at the tested concentration of 50 μg/plate in absence of metabolic activation system in all the tester strains. Normal growth was observed in all tester strains at the tested concentrations of 1.5 – 15 μg/plate in the absence of the metabolic activation system and 1.5 – 150 μg/plate in the presence of the metabolic activation system.

No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

From the results of the Initial Toxicity-Mutation Assay, 150 μg/plate was selected in absence of metabolic activation system and 600 μg/plate was selected in the presence of metabolic activation system for all tester strains, being the highest concentration for the Confirmatory Mutation Assay.

In the Confirmatory Mutation Assay, bacterial cultures were exposed to Butyl Phenyl Ether at concentrations of 2.3, 4.7, 9.4, 18.8, 37.5, 75, and 150 μg/plate in the absence of metabolic activation system and 9.4, 18.8, 37.5, 75, 150, 300, and 600 μg/plate in the presence of metabolic activation system (10% v/v S9 mix) for all tester strains (three plates/concentration). Complete inhibition of background lawn (with 100% reduction in revertant colonies) was observed at the tested concentration of 150 μg/plate in the absence of metabolic activation system and at the tested concentration of 600 μg/plate in the presence of metabolic activation system in all the tester strains. Partial inhibition of background lawn with reduction in revertant colonies (42-54%) was observed at the tested concentration of 75 μg/plate in the absence of metabolic activation system in all tester strains. Partial inhibition of background lawn with reduction in revertant colonies (45-52%) was observed at 300 μg/plate in presence of metabolic activation system in all tester strains. Normal growth was observed at other tested concentrations in the absence and presence of metabolic activation system in all tester strains. No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

The Butyl Phenyl Ether stock concentrations 23, 47, 94, 188, 375, 750, 1500, 3000 and 6000 μg/mL was found to be within acceptable range of ± 15% of nominal concentrations during the Confirmatory Mutation Assay. The 0 hour concentrations of butyl phenyl ether in the dose formulation were found to be 98.4%, 92.1%, 97.0%, 91.0%, 94.4%, 95.0%, 92.8%, 94.3% and 90.5% of dose level 23 μg/mL, 47 μg/mL, 94 μg/mL, 188 μg/mL, 375 μg/mL, 750 μg/mL, 1500 μg/mL, 3000 μg/mL and 6000 μg/mL, respectively. The concentrations of butyl phenyl ether in the dose formulation after 4 hours were found to be 105% and 106% of the 0 hour concentrations of dose level T1 (23 μg/mL) and T7 (6000 μg/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, Butyl Phenyl Ether is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2uvrA (pKM101).

Mammalian Clastogenicity

Butyl phenyl ether (Butoxybenzene) 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 1502.0 μg butyl phenyl ether per ml of culture medium. The highest concentration was based on the limit concentration specified in the guideline (10 mM). The duration of treatment was 4 hours without and with S9 and 24 hours without S9. The analytically determined concentrations of butyl phenyl ether in the dose preparations ranged from 88.7 to 136.0% of the targeted values. Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon cytotoxicity of the test material. In the initial cytogenetic assay cultures treated for 4 hours with targeted concentrations of 0.0 (vehicle control), 2.1, 18.5, and 55.6 μg/ml in the presence of S9 were selected for determining the incidence of chromosome aberrations based upon cytotoxicity. At the highest dose (55.6 μg/ml) a statistically significant increase in the frequency of aberrant cells was observed (1.3% in the vehicle control vs. 7.7% at 55.6 μg/ml), and the observed increase was dose-dependent. This frequency of aberrant cells at 55.6 μg/ml was outside of the laboratory’s historical control range. In the initial assay the 4 and 24 hour treatment conditions in the absence of S9 the guideline required level of cytotoxicity (60 + 10%) was not achieved, therefore, these cultures were not evaluated for chromosome aberration frequency.

In the repeat assay, cultures were treated with concentrations ranging from 0.0 (vehicle control) to 160 μg/ml for 4 hours, in the absence and presence of S9. Based upon the mitotic indices, cultures treated in the absence of S9, with targeted concentrations of 0 (vehicle control), 50.0, 70.0, and 90.0 μg/ml and cultures treated in the presence of S9, with targeted concentrations of 0.0 (vehicle control), 2.0, 50.0, and 60.0 μg/ml were selected for determining the incidence of chromosomal aberrations.

There were no statistically significant increases in the frequencies of cells with aberrations in the cultures treated for 4 hours in the absence of S9 when compared to the vehicle control cultures. There was a significant increase in the frequency of cells with aberrations in the presence of S9, with 0.7% in the vehicle control and 4.7% and 7.0% at the highest two concentrations (i.e., 50.0 and 60.0 μg/ml), respectively. The 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. The vehicle and positive controls in all assays were within the acceptable historical ranges and fulfilled the requirements for a valid assay. As a result of the clear positive response observed in the 4 hour treatment (with S9) the 24 hour treatment condition was not evaluated.

Based upon these results, butyl phenyl ether was considered to be positive for clastogenicity in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

In vivo clastogenicity

To follow up the positive findings from the in vitro rat chromosome aberration assay, a rat bone marrow micronucleus assay was incorporated into a repeated dose/reproductive screening assay (OECD 422) that was being performed to meet the requirements of the REACH submission.

Incorporating this assay into a repeated dose toxicity study allowed the assessment of clastogenicity without the need for a separate stand-alone study, reducing animal usage while delivering data in order to form a judgement of in vivo clastogenicity.

In the OECD 422 study, male and female Sprague Dawley rats were dosed daily via gavage with either 0, 30, 100 or 350 mg/kg bw/day butyl phenyl ether in corn oil. On study day 36 at the necropsy for males, 5 males from each group were selected for harvesting of the bone marrow. In addition, 5 females from each dose group were selected at study termination for bone marrow harvesting. The positive control group (5 males) were dosed once with cyclophosphamide (20 mg/kg bw, via gavage) 24 hours prior to scheduled necropsy, at which point bone marrow was harvested for the micronucleus assessment. Since both males and females had a similar response to treatment with butyl phenyl ether only the male bone marrow samples were assessed for micronuclei formation.

Whenever possible, a minimum of 4000 polychromatic erythrocytes (PCE) were examined from each animal and the number of MN-PCE were recorded.  The proportion of PCE among total erythrocytes (PCE and normal chromatic erythrocytes (NCE)) in the bone marrow were determined by examining at least 500 erythrocytes.  The percentage of PCE was expressed as PCE /PCE+NCE x 100.

The %PCE observed in the groups treated with butyl phenyl ether was not significantly different from the vehicle control group. As expected, the %PCE of the positive control group was significantly lower than that of the vehicle control group. The MN-PCE frequency (/1000) in the vehicle control group was 3.30 ± 1.10 (mean ± SD). The MN-PCE frequencies in the 30, 100, and 350 mg/kg bw/day treatment groups were 3.30 ± 1.32, 3.69 ± 0.85, and 3.72 ± 1.01, respectively. There were no significant differences in MN-PCE frequency among the groups treated with butyl phenyl ether and the vehicle control group. The adequacy of the experimental conditions for the detection of induced micronuclei was confirmed with the observation of a significant increase in the frequencies of MN-PCE in the positive control group (35.65 ± 3.74).

The treatment-related effects on liver and urinary bladder (see below) demonstrated the systematic distribution of the test material and/or its metabolites after oral gavage. The treatment-related hematologic effects (e.g., statistically significant higher mean reticulocyte count in the 350 mg/kg/day males) further demonstrated the adequate bone marrow exposure to the test material and/or its metabolites. Therefore, butyl phenyl ether was negative in this bone marrow erythrocyte MNT under the experimental conditions used.

Summary:

Butyl phenyl ether was clearly negative for mutagenicity in bacteria in the presence and absence of metabolic activation. However, in the presence of metabolic activation it produced an increase in the percentage of aberrations in rat lymphocytes incubated with butyl phenyl ether for 4 hours. Consequently no further assessment of in vitro genotoxicity was performed. Since a repeated dose toxicity study was scheduled to meet the REACH requirements, an in vivo micronucleus assay was incorporated into the protocol to provide an in vivo assessment of clastogenic potential.

In this assay there was clear evidence of systemic exposure and systemic toxicity, and this is consistent with the modelled toxicokinetic data showing >99% systemic bioavailability following oral exposure and a low volume of distribution (6.7 l/Kg) indicating that there is little tissue partitioning of this substance in the body.

Based on the presence of toxicity and the absence of an increase in micronucleated PCEs in all treated groups, it is concluded that butyl phenyl ether is not an in vivo clastogen. No further in vivo genotoxicity testing is therefore proposed.

Justification for classification or non-classification

Classification criteria for mutagenicity are not met.

The substance does not meet the classification criteria as described in regulation EC 1272/2008 or its accompanying guidance