5-bromo-1,3-dichloro-2-fluorobenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

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:

5-bromo-1,3-dichloro-2-fluorobenzene

EC Number:

640-454-2

Cas Number:

17318-08-0

Molecular formula:

C6H2 BrCl2F

IUPAC Name:

5-bromo-1,3-dichloro-2-fluorobenzene

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Wistar rats (RjHan:WI; weight approx. 220 – 320 g, Janvier Labs, 53941 Saint-Berthevin Cedex, France) induced by peroral administration of 80 mg/kg b.w. phenobarbital (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) and by peroral administrations of ß-naphthoflavone (Acros Organics, 2440 Geel, Belgium) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene (Appendix 3).
The protein concentration in the S9 preparation was 32.6 mg/mL (lot no. 010617E) in the pre-experiment / Experiment I and 30.4 mg/mL (lot no. 261017K) in Experiment II.
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2 33 mM KCl 5 mM Glucose-6-phosphate 4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al..

Test concentrations with justification for top dose:

Pre-experiment / Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
Strains TA 100 and WP2 pKM101: 0.3; 1, 3, 10; 33; 100; 333; and 1000 µg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Based on the observed cytotoxicity the maximal concentration was 5000 μg/plate for strains TA 1535, TA 1537, TA 98, and WP2 uvrA pKM101, respectively 1000 μg/plate for strains TA 100 and WP2 pKM101.

Vehicle / solvent:

dimethylsulfoxide (DMSO, purity 99%)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

The thawed bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing nutrient medium (50 mL). A solution of ampicillin (50 μL, 25 μg/mL) was added to the strains TA 98, TA 100, WP2 uvrA pKM101, and WP2 pKM101. This nutrient medium contains per liter:
8 g Nutrient Broth (MERCK, 64293 Darmstadt, Germany) 5 g NaCl (MERCK, 64293 Darmstadt, Germany)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10^8-10^9 cells/mL).
Plates with selective agar (without Histidine/Tryptophan) were used.
The overlay agar contained per litre:
for Salmonella strains:
7.0 g Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine×HCl×H2O*
12.2 mg Biotin*
for Escherichia coli strains:
7.0 g Agar Agar*
6.0 g NaCl*
10.2 mg Tryptophan*
* (MERCK, 64293 Darmstadt, Germany)
Sterilisations were performed at 121 °C in an autoclave.
To evaluate the cytotoxicity of the test substance a pre-experiment was performed with all strains. Eight concentrations were tested for cytotoxicity and mutation induction each with three replicate plates. The experimental conditions in this pre-experiment are described in section 3.7 (plate incorporation test).
Cytotoxicity of the test substance results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as the Main Experiment I since the criteria mentioned in Section 3.8.2: Acceptability of the Assay were met.
For each strain and concentration including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each concentration, solvent (negative control) or reference mutagen solution (positive control),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer* (for test without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains; OD = 0.9 - 1.2; wavelength = 500 nm; approx. 8x108 cells/mL),
2000 μL Overlay agar
For the pre-incubation method test solution (100 μL) (solvent or reference mutagen solution (positive control)), S9 mix / S9 mix substitution buffer* (500 μL) and bacteria suspension (100 μL) were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation overlay agar (2.0 mL, 45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for 72 hours at 37°C in the dark, plates were then stored at 4°C until counted. (6).
In parallel to each test a sterile control of the test substance was performed and documented in the raw data. Therefore, stock solution (100 μL) and S9 mix / S9 mix substitution buffer* (500 μL) were mixed with overlay agar (2.0 mL) and poured on minimal agar plates.
* Substitution buffer: 7 parts of the 100 mM sodium-ortho-phosphate-buffer pH 7.4 with 3 parts of KCl solution 0.15 M

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants of twice or above the spontaneous mutation rate of the corresponding solvent control is observed (1).
A concentration dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration (6).
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Plates incubated with the test item showed reduced background growth at the following concentrations (μg/plate)

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	1000 – 5000	1000 – 5000	1000 – 5000	1000 – 5000
TA 1537	1000 – 5000	1000 – 5000	100 – 5000	333 – 5000
TA 98	1000 – 5000	2500 – 5000	333 – 5000	1000 – 5000
TA 100	2500 – 5000	333 – 5000	1000	333 – 1000
WP2 pKM101	333 – 5000	1000 – 5000	/	1000
WP2 uvrA pKM101	2500 – 5000	1000 – 5000	2500 – 5000	1000 – 5000

/ = normal background growth

Table 2: Cytotoxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate)

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	5000	2500 – 5000	2500 – 5000	2500 – 5000
TA 98	2500 – 5000	/	1000 – 5000	2500
TA 100	333 – 5000	333 – 5000	100 – 1000	333 – 1000
WP2 pKM101	333 – 5000	2500 – 5000	100 – 1000	1000
WP2uvrApKM101	/	5000	333 – 5000	1000 – 5000

/ = no cytotoxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

Table 3: Summary of results of pre-experiment/experiment I

Metabolic Activation	Test Group	Concen-tration (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO			12 ± 2	8 ± 3	26 ± 6	168 ± 8	233 ± 12	249 ± 7
	Untreated			14 ± 3	12 ± 5	23 ± 3	190 ± 6	232 ± 27	290 ± 14
	CA5528	3 µg		15 ± 5	9 ± 3	22 ± 2	151 ± 19	213 ± 15	239 ± 19
	tech.	10 µg		13 ± 4	11 ± 1	31 ± 3	152 ± 4	216 ± 5	252 ± 13
		33 µg		11 ± 1	10 ± 2	33 ± 7	145 ± 20	223 ± 13	250 ± 5
		100 µg		11 ± 1	10 ± 1	22 ± 5	85 ± 6	172 ± 13	215 ± 30
		333 µg		12 ± 4	8 ± 2	25 ± 5	52 ± 9	68 ± 8^R	147 ± 6
		1000 µg		8 ± 3^R	7 ± 2^M R	15 ± 1^M R	41 ± 6	61 ± 4^R	136 ± 10
		2500 µg		10 ± 1^R	5 ± 2^M R	12 ± 3^M R	44 ± 7^R	58 ± 2^R	134 ± 3^R
		5000 µg		14 ± 3^R	3 ± 1^M R	9 ± 2^M R	36 ± 6^M R	54 ± 7^R	123 ± 13^R
	NaN3	10 µg		1228 ± 25			2121 ± 130
	4-NOPD	10 µg				323 ± 27
	4-NOPD	50 µg			125 ± 9
	MMS	2.0 µL						3741 ± 24	2926 ± 12
With Activation	DMSO			13 ± 4	11 ± 3	30 ± 2	143 ± 11	215 ± 31	277 ± 20
	Untreated			14 ± 2	15 ± 3	41 ± 7	166 ± 17	259 ± 38	338 ± 48
	CA5528	3 µg		11 ± 2	10 ± 1	38 ± 6	126 ± 10	229 ± 4	326 ± 25
	tech.	10 µg		12 ± 2	14 ± 3	37 ± 3	148 ± 27	220 ± 7	261 ± 39
		33 µg		18 ± 3	10 ± 1	41 ± 7	160 ± 8	203 ± 28	240 ± 12
		100 µg		12 ± 4	12 ± 3	36 ± 9	159 ± 17	231 ± 13	265 ± 32
		333 µg		15 ± 4	15 ± 1	37 ± 9	47 ± 9^R	196 ± 26	296 ± 22
		1000 µg		12 ± 2^R	9 ± 2^M R	41 ± 5	46 ± 4^R	105 ± 18^R	157 ± 16^R
		2500 µg		11 ± 2^M R	5 ± 2^M R	33 ± 4^M R	31 ± 4^M R	76 ± 11^R	146 ± 16^R
		5000 µg		7 ± 2^M R	2 ± 1^M R	23 ± 5^M R	24 ± 4^M R	38 ± 11^M R	80 ± 10^M R
	2-AA	2.5 µg		543 ± 28	106 ± 7	4009 ± 62	3596 ± 184
	2-AA	10.0 µg						1212 ± 29	2025 ± 6

Table 4: Summary of results of experiment II

Metabolic Activation	Test Group	Concen-tration (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO			8 ± 3	11 ± 1	39 ± 2	152 ± 18	230 ± 8	346 ± 28
	Untreated			8 ± 2	16 ± 5	37 ± 3	204 ± 19	286 ± 20	384 ± 35
	CA5528	0.3 µg					152 ± 14	206 ± 8
	tech.	1 µg					162 ± 4	209 ± 2
		3 µg		6 ± 1	12 ± 4	32 ± 10	153 ± 12	228 ± 22	384 ± 7
		10 µg		8 ± 2	11 ± 3	35 ± 8	159 ± 17	223 ± 11	388 ± 36
		33 µg		6 ± 2	11 ± 4	27 ± 7	128 ± 22	207 ± 4	285 ± 34
		100 µg		10 ± 1	11 ± 1^R	19 ± 3	49 ± 6	95 ± 14	180 ± 2
		333 µg		8 ± 3	6 ± 2^M R	20 ± 6^R	37 ± 6	51 ± 7	124 ± 14
		1000 µg		9 ± 3^R	6 ± 2^M R	12 ± 4^M R	38 ± 8^R	48 ± 7	109 ± 17
		2500 µg		10 ± 2^R	5 ± 2^M R	9 ± 3^M R			101 ± 10^R
		5000 µg		6 ± 2^M R	3 ± 1^M R	1 ± 1^M R			90 ± 7^R
	NaN3	10 µg		1266 ± 5			2109 ± 114
	4-NOPD	10 µg				351 ± 22
	4-NOPD	50 µg			181 ± 13
	MMS	2.0 µL						4237 ± 145	3630 ± 101
With Activation	DMSO			12 ± 3	16 ± 5	43 ± 6	132 ± 12	229 ± 4	467 ± 11
	Untreated			8 ± 2	17 ± 5	42 ± 1	195 ± 14	282 ± 13	466 ± 16
	CA5528	0.3 µg					126 ± 22	250 ± 15
	tech.	1 µg					151 ± 16	257 ± 20
		3 µg		10 ± 1	16 ± 5	46 ± 5	143 ± 6	216 ± 7	504 ± 11
		10 µg		13 ± 2	19 ± 4	38 ± 5	146 ± 15	218 ± 17	488 ± 4
		33 µg		10 ± 1	19 ± 3	36 ± 6	111 ± 15	232 ± 36	448 ± 47
		100 µg		13 ± 2	19 ± 3	46 ± 6	109 ± 17	258 ± 16	404 ± 43
		333 µg		10 ± 3	21 ± 5^R	37 ± 0	32 ± 3^R	119 ± 17	226 ± 32
		1000 µg		9 ± 3^R	19 ± 2^M R	31 ± 7^R	25 ± 7^R M	58 ± 12^R	131 ± 5^R
		2500 µg		6 ± 2^M R	6 ± 2^M R	18 ± 3^M R			70 ± 4^R M
		5000 µg		19 ± 6^M R	1 ± 1^M R	34 ± 12^M R			53 ± 5^M R
	2-AA	2.5 µg		440 ± 26	144 ± 26	4137 ± 266	3572 ± 33
	2-AA	10.0 µg						1173 ± 117	1933 ± 96

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	R M	Reduced background growth Manual count

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity tests and under the experimental conditions reported, the substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the substance to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA pKM101 and WP2 pKM101.

The plates incubated with the test item showed reduced background growth in all strains used with and without S9 mix.

Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains, except of strain TA 1535.

No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).  There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls, which showed a distinct increase of induced revertant colonies demonstrating the correct performance of the assay.