Boronic acid, B-(4-chloro-2-fluoro-3-methoxyphenyl)-

Genetic toxicity: in vitro

Administrative data

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:

8 May 2012 to 1 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

METI Reverse Mutagenicity Test on Bacteria, Methods of Testing New Chemical Substances (Section 5.1-1 to 5.1-11): Japanese Act on Evaluation of Chemical Substances and Regulation of Their Manufacture, Act No. 117, Ministerial Ordinance No.1-3 (April 2004)

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:

944127-07-1

IUPAC Name:

944127-07-1

Test material form:

other: solid (unspecified)

Details on test material:

- Appearance: White Solid
- Storage conditions of the test material: Ambient (18 to 36 °C)

Method

Target gene:

Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: S. typhimurium: all strains possess rfa and uvrB; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA mutation and the R-factor plasmid pKM101.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from induced rat liver

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Confirmatory mutation assay: 0, 93.8, 187.5, 375, 750, 1500, 3000 and 5000 µg/plate for strain TA 100
Confirmatory mutation assay: 0, 187.5, 375, 750, 1500, 3000 and 5000 µg/plate for strains TA 98, TA 1535, TA 1537 and WP2uvrA

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO is one of the organic vehicles compatible with this test system. The test material was found to form a solution in DMSO at 500 mg/mL and was stable at 15 and 50000 μg/mL after 4 hours.

Controls

Untreated negative controls:

no

Remarks:

Sterility control plates were observed for microbial colonies. Viable count plates were observed to determine the number of colony forming units per mL of each bacterial suspension.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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:

METHOD OF APPLICATION: pre-incubation
100 µL of the appropriate bacterial culture, 100 µL of the test material or control material solution and 500 µL of the S9 mix or PBS were transferred into sterile test tubes and were kept in an incubator shaker for approximately 20 ± 2 minutes at 37 ± 1 °C. 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.

DURATION
- Exposure duration: 67 hours at 37 ± 1 °C under yellow light

NUMBER OF REPLICATIONS
- Initial toxicity-mutation assay: duplicate
- Confirmatory mutation assay: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth

Evaluation criteria:

The conditions necessary for determining a positive result are as follows:
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 material either in the absence or presence of the metabolic activation system.

- Strains TA98, TA1535, and TA1537
Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0 times the mean vehicle control value.

- Strain TA100 and WP2uvrA
Data sets will be 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 does not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) will not be evaluated as positive.

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium TA98, TA1535, TA1537 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: Viable counts of all the tester strains were within the required range of 1 to 2 x10⁹ CFU/mL. The most concentrated test material dilution, the Sham (PBS) and S9 mixes were found to be sterile.

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: Viable counts of all the tester strains were within the required range of 1 to 2 x10⁹ CFU/mL. The most concentrated test material dilution, the Sham (PBS) and S9 mixes were found to be sterile.

Positive controls validity:

valid

Additional information on results:

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 material did not cause any precipitation on the basal agar plates up to 5000 µg/plate. No toxicity was observed up to up to 1500 µg/plate as the intensity of the bacterial background lawn was comparable to the vehicle control in the presence and absence of metabolic activation. However, at the top dose of 5000 µg/plate, there was slight to moderate thinning of the bacterial background lawn in the presence and absence of metabolic activation.
The test material was found to be toxic to all the five tester strains at the top dose of 5000 μg/mL when compared to the vehicle control, both in the presence and absence of metabolic activation. For the strain TA 100, there was a greater than 2-fold increase in the mean number of revertant colonies compared to the vehicle control at the dose of 1500 μg/plate both in the presence and 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
Summary results from the confirmatory toxicity-mutation assay are presented in Table 1.
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 material did not cause any precipitation on the basal agar plates up to 5000 µg/plate. The toxicity profile of the test material varied with the tester strains in the confirmatory mutation assay.
Strains TA 98, TA 1535 and TA 1537 did not exhibit any toxicity up to 1500 µg/plate as the intensity of the bacterial background lawn was comparable to the vehicle control in the presence and absence of metabolic activation. However, TA 98 exhibited slight thinning at 3000 µg/plate, and moderate thinning of the background lawn at the top dose of 5000 µg/plate, in the presence and absence of metabolic activation. Similarly, strains TA 1535 and TA 1537 exhibited moderate thinning of the background lawn at and above 3000 µg/plate, in the presence and absence of metabolic activation.
Strains TA 100 and WP2uvrA (pKM101) did not exhibit any toxicity up to 3000 µg/plate as the intensity of the bacterial background lawn was comparable to the vehicle control in the presence and absence of metabolic activation. However, at the top dose of 5000 µg/plate, there was slight thinning of the bacterial background lawn in the presence and absence of metabolic activation for these 2 tester strains.

There was no positive mutagenic response observed in the strains TA 98, TA 1535, TA 1537 and WP2uvrA (pKM101) in any of the tested doses either in the presence or in the absence of metabolic activation. However, there was ≥ 2-fold increase in the mean number of revertant colonies for the strain TA 100 at 1500 and 3000 μg/plate in the presence of metabolic activation and at 750, 1500 and 3000 μg/plate in the absence of metabolic activation compared to the vehicle control.

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.

S-9 HOMOGENATE
The S-9 homogenate was found to be sterile, active and the protein content of the S-9 homogenate was 26.6 mg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary Results of the Confirmatory Mutation Assay

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
- - - - - - - -	Solvent 93.8 187.5 375 750 1500 3000 5000	116 119 135 199 231*** 256*** 287*** 52*	14 - 13 12 11 10 4* 2*	133 - 133 139 145 161 175 65*	26 - 25 24 32 30 15** 4*	11 - 11 10 9 8 3* 2*
+ + + + + + + +	Solvent 93.8 187.5 375 750 1500 3000 5000	105 111 124 139 180 255*** 266*** 57**	16 - 16 13 14 11 3* 2*	141 - 154 168 168 178 192 80**	27 - 24 30 33 38 15** 6*	11 - 10 9 9 9 2* 2*
Positive Controls
-	Name	SA	SA	4NQO	2NF	9AA
	Concentration (µg/plate)	1	1	4	2	50
	Mean no. colonies/plate	554	127	589	236	101
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	4	4	30	4	4
	Mean no. colonies/plate	871	137	556	542	109

*Cytotoxicity observed as a >50 % reduction in revertants compared to the vehicle control

**Cytotoxicity observed as a reduced background lawn

***Mutagenic (positive) result at ≥2-fold increase in revertants as compared to the vehicle control

SA = sodium azide

4NQO = 4-nitroquinoline-1-oxide

2NF = 2-nitrofluorene

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive in strain TA 100 with and without metabolic activation

Under the conditions of this study, the test material is considered to be mutagenic for the S. typhimurium tester strain TA 100 in both the presence and absence of S9 activation.

Executive summary:

The test material was assessed for mutagenic potential in the bacterial reverse mutation assay in accordance with the standardised guidelines OECD 471, EU Method B.13/14, USA EPA OPPTS 870.5100 and the Japanese METI Reverse Mutagenicity Test on Bacteria under GLP conditions.

The study was conducted using TA 98, TA 100, TA 1535 and TA 1537 strains of Salmonella typhimurium and the WP2uvrA (pKM101) strain of Escherichia coli in two phases. In the first phase, an initial toxicity-mutation test was performed. The second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test material in dimethyl sulphoxide in the presence and absence of a metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver) using a pre-incubation procedure.

In the initial toxicity-mutation assay, cultures were exposed to the test material in duplicate at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, along with the vehicle and appropriate positive controls.

The test material did not cause any precipitation on the basal agar plates up to 5000 μg/plate. No toxicity was observed up to 1500 μg/plate. However, the test material was found to be toxic to all five tester strains at the top dose of 5000 μg/mL, both in the presence and absence of metabolic activation when compared to the vehicle control.

There was no positive mutagenic response observed in the strains TA 98, TA 1535, TA 1537 and WP2uvrA (pKM101) in any of the tested doses either in the presence or in the absence of metabolic activation.

However, TA 100 resulted in a greater than 2-fold increase in the mean number of revertant colonies compared to the vehicle control at 1500 μg/plate both in the presence and absence of metabolic activation.

Based on these initial findings, in the confirmatory mutation assay, cultures were exposed in triplicate at 93.8, 187.5, 375, 750, 1500, 3000 and 5000 μg/plate test doses for the strain TA 100 and 187.5, 375, 750, 1500, 3000 and 5000 μg/plate test doses for the strains TA 98, TA 1535, TA 1537 and 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 material did not cause any precipitation on the basal agar plates up to 5000 μg/plate. The toxicity profile of the test material varied with the tester strains in the confirmatory mutation assay. Strains TA 98, TA 1535 and TA 1537 exhibited toxicity as evidenced by the intensity of the bacterial background lawn at concentrations of 1500 µg/plate and higher, in the presence and absence of metabolic activation. Strains TA 100 and WP2uvrA (pKM101) exhibited toxicity at concentrations of 3000 μg/plate and higher, in the presence and absence of metabolic activation.

There was no positive mutagenic response observed in the strains TA 98, TA 1535, TA 1537 and WP2uvrA (pKM101) in any of the tested doses either in the presence or in the absence of metabolic activation. However, there was a 2-fold increase in the mean number of revertant colonies for the strain TA 100 at 1500 and 3000 μg/plate in the presence of metabolic activation and at 750, 1500 and 3000 μg/plate test doses in the absence of metabolic activation compared to the vehicle control.

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.

Under the conditions of this study, the test material is considered to be mutagenic for the S. typhimurium tester strain TA 100 in both the presence and absence of S9 activation.