N-butyl phenyl ether

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

N-butyl phenyl ether

EC Number:

214-426-1

EC Name:

N-butyl phenyl ether

Cas Number:

1126-79-0

Molecular formula:

C10H14O

IUPAC Name:

butoxybenzene

Details on test material:

Test Material Name: Butyl phenyl ether
Chemical Name: Butoxy benzene
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).

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

Method

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

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).