1,2-dichloro-4-(trichloromethyl)benzene

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:

September - November 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: in accordance with guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

1,2-dichloro-4-(trichloromethyl)benzene

EC Number:

235-869-7

EC Name:

1,2-dichloro-4-(trichloromethyl)benzene

Cas Number:

13014-24-9

Molecular formula:

C7H3Cl5

IUPAC Name:

1,2-dichloro-4-(trichloromethyl)benzene

Details on test material:

- Name of test material (as cited in study report): 3,4-Dichloro-benzotrichloride
- Substance type: intermediate for synthesis
- Physical state: Liquid/Colourless to slightly yellow/characteristic aromatic
- Analytical purity: 99.8% (as per Certificate of Analysis)
- Purity test date: March 2010
- Lot/batch No.: 03/10
- Expiration date of the lot/batch: March 2012
- Storage condition of test material: stored in its original container, at room temperature away from heat.

Method

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:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

A volume of 0.1 mL of the test substance stock solution of the lowest concentration was added aseptically to 2 mL of top agar, mixed well and this mixture was poured onto MGA plates.

Vehicle / solvent:

A volume of 0.1 mL of dimethyl sulfoxide used as vehicle was added aseptically to 2 mL of top agar, mixed well and this mixture was poured onto MGA plates.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

75.0 µg/plate, in absence of metabolic activation Migrated to IUCLID6: for TA1537

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

0.5 µg/plate for TA1535 and 5.0 µg/plate for TA100, in absence of metabolic activation Migrated to IUCLID6: for TA1535 and TA100

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

7.5 µg/plate, in absence of metabolic activation Migrated to IUCLID6: for TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.5 µg/plate, in absence of metabolic activation Migrated to IUCLID6: for TA102

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene for TA1535, TA1537, TA102

Remarks:

10.0 µg/plate, with metabolic activation

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene for TA 98 and TA100

Remarks:

5.0 µg/plate, with metabolic activation

Details on test system and experimental conditions:

The Salmonella typhimurium strains used in this study were mutants derived from Salmonella typhimurium LT2. The strains used in the study were obtained from Xenometrix GmbH, Gewerbestraesse 25, CH 4123 Allschwil, Switzerland.

Metabolic Activation System (S9 Fraction and S9 Mix):
Unlike mammals, bacteria lack the necessary oxidative enzyme systems for metabolising foreign compounds to electrophilic metabolites capable of reacting with DNA. Sometimes these foreign compounds, when reacting with a mammalian enzyme system, yield mutagenic metabolic products. In order to test these indirectly acting mutagens, a metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction is used. The S9 fraction is buffered and supplemented with the essential co-factors  NADP and Glucose-6- phosphate to form the "S9 mix". This mix is added to the top agar in this assay. The S9 fraction prepared at Section Mutagenicity, Department of Toxicology, JRF was used in the study.

Composition of Co-factor Mix :
D- Glucose –6- phosphate:0.80 g
beta Nicotinamide adenine dinucleotide phosphate (beta NADP): 1.75 g
Magnesium chloride:0.90 g
Potassium chloride:1.35 g
Sodium phosphate, dibasic:6.40 g
Sodium phosphate, monobasic:1.40 g
Distilled water :450 mL
The prepared co-factor mix was dispensed to suitable volumes and stored below 0 °C.

Composition of Top Agar and Minimal Glucose Agar:
Top Agar with 0.5mM histidine/biotin:
Bacto Agar :0.6 g
Sodium chloride (NaCl) :0.6 g
0.5 mM histidine/biotin solution :10 mL
Distilled water :90 mL
Minimal Glucose Agar plates:
Bacto Agar :7.5 g
Distilled water :450 mL
50X Vogal Bonner medium:10 mL
20% Glucose solution:25 mL

CYTOTOXICITY TEST
Before commencing the mutagenicity study, 3,4-dichloro-benzotrichloride was tested for possible cytotoxicity, if any, to Salmonella typhimurium tester strain TA100. The experiment was conducted both in the absence and presence of metabolic activation system (5% v/v S9 mix).
A stock solution of 50 µL/mL of 3,4-dichloro-benzotrichloride was prepared by dissolving 100 µL in 2 mL of dimethyl sulfoxide (Stock A). Further stock solutions of concentrations, viz., 25 (Stock B), 12.5 (Stock C), 6.25 (Stock D), 3.125 (Stock E), 1.5625 (Stock F), 0.7813 (Stock G), 0.3906 (Stock H), 0.1953 (Stock I) and 0.0977 µL/mL (Stock J) were prepared from the first stock solution. Ten different concentrations, viz, 0.0098, 0.0195, 0.0391, 0.0781, 0.1563, 0.3125, 0.625, 1.25, 2.5 and 5.0 µL/plate were tested for cytotoxicity (The dose values are represented in the report to four decimal places). Volumes of 100 µL of relevant stock solutions A - J were used to obtain the required test concentrations for treatment both in the absence and presence (5% v/v S9 mix) of the metabolic activation system.
Tubes containing 2 mL of molten top agar with 0.5 mM histidine/biotin were maintained at 45 ± 2 °C. A volume of 100 µL of the relevant stock solution of the test substance and dimethyl sulfoxide were used for treatment, as a negative control, respectively.
A volume of 500 µL of 5% v/v S9 mix was added to one of the sets and 500 µL of 0.2 M phosphate buffer was added to the second set. Finally 100 µL of bacterial culture was added to the tubes and mixed. Cultures used were checked for cell viability prior to testing . This treatment mixture was poured on MGA plates and allowed to solidify. Triplicate sets were maintained for each concentration of 3,4-dichloro-benzotrichloride and negative control. The petriplates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the state of background bacterial growth inhibition and reduction in number of colonies.

MUTAGENICITY TEST
The mutagenicity test was conducted as two independent experiments. In both the trials, the treatment was performed both in the absence and presence of metabolic activation system (5% v/v and 10% v/v S9 mix in Trial I and II, respectively). The treatments were performed by the plate incorporation technique as described in the cytotoxicity test. Plates were maintained in triplicate for each test concentration of 3,4-dichloro-benzotrichloride, negative and positive controls.
Trial I
The tester strains were exposed to concentrations of 0.0025, 0.005, 0.01, 0.02, 0.04 and 0.08 µL/plate both in the absence and presence (5% v/v S9 mix) of metabolic activation system for trial I (factor 2). The stock solution (Stock A) of the test substance was prepared by dissolving 80 µL of 3,4-dichloro-benzotrichloride in dimethyl sulfoxide and made up to 10 mL (8 µL/mL). For the treatment, a volume of 1 mL of Stock A was added to 9 mL dimethyl sulfoxide to obtain a concentration of 0.8 µL/mL (Stock B). A volume of 5 mL of Stock B was added to 5 mL dimethyl sulfoxide to obtain a concentration of 0.4 µL/mL (Stock C). Further stock solutions viz., 0.2 (Stock D), 0.1 (Stock E), 0.05 (Stock F), and 0.025 (Stock G) were prepared by serial two fold dilution. Volumes of 100 µL of relevant stock solutions B - G were used to obtain the required test concentrations for treatment both in the absence and presence (5% v/v S9 mix) of the metabolic activation system.
Trial II
A second trial was conducted to confirm the negative results obtained in Trial I. In Trial II, the concentration spacing was modified using a factor of 2.5 and the concentration of S9 mix was increased to 10 % v/v. The highest concentration being 0.08 µL/plate, five lower concentrations viz., 0.032, 0.0128, 0.0051, 0.0020 and 0.0008 µL/plate were tested both in the absence and presence (10% v/v S9 mix) of metabolic activation system. Appropriate stock solutions of test substance were prepared prior to treatment. Volumes of 100 µL of these stock solutions were used to obtain the required test concentrations. Plates were maintained in triplicate for each test concentration of 3,4-dichloro-benzotrichloride, negative and positive controls during both the trials. The number of revertant colonies was recorded after 48 h incubation period.
Treatment with 2-aminoanthracene in the absence of metabolic activation was performed for tester strain TA100 in both the trials to demonstrate the efficiency of the S9 fraction used in the study.

Evaluation criteria:

Before assay data are evaluated, criteria for a valid assay have to be met. The following criteria are used to determine a valid assay: tester strain integrity, characteristic number of spontaneous revertants, tester strain culture density, positive control values in the absence of metabolic activation, positive control values in the presence of metabolic activation, cytotoxicity.
Once criteria for a valid assay have been met, responses observed in the assay are evaluated. The conditions necessary for determining a positive result are there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test substance either in the absence or presence of the metabolic activation system.
Strains TA98, TA1535, and TA1537
Data sets are 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 TA102
Data sets are 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.
Statistical analysis was used as an aid in the evaluation of dose response.
A response that does not meet all three of the above criteria (magnitude, concentration- responsiveness, reproducibility) was not evaluated as positive. Negative results obtained in the first trial were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing and metabolic activation.

Statistics:

Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

5% v/v S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Negative Control:
The results of the study indicate that the values of negative control in all strains are within limits.

Positive Controls:
2-Aminoanthracene was used as the positive control in the presence of metabolic activation for all the tester strains during both the trials. Large historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation. The batch of S9 used in this study was characterised with benzo(a)pyrene that requires metabolic activation by microsomal enzymes. Benzo(a)pyrene exhibited clear increase in the number of revertants when compared with the concurrent negative control which demonstrated the efficiency of S9 used in this study.
Positive controls exhibited a clear increase in the number of revertants when compared with the concurrent negative and vehicle controls. This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.
Increase in revertants were not observed in tester strain TA100 (Trial I and II) treated with 2-aminoanthracene in the absence of metabolic activation but clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Cytotoxicity Test:
Cytotoxicity is characterised by inhibition of the background bacterial lawn and reduction in the number of colonies. Inhibition of background bacterial lawn was observed at the concentrations of 0.1563, 0.3125, 0.625, 1.25, 2.5 and 5.0 µL/plate both in the absence and presence of the metabolic activation system and at the concentration of 0.0781 µL/plate in the absence of metabolic activation system. Partial inhibition of background bacterial lawn was observed at the concentration of 0.0391 µL/plate both in the absence and presence of metabolic activation system and at 0.0781 µL/plate in the presence of metabolic activation system. Reduction in the number of colonies was observed at the concentration of 0.0781 µL/plate and micro-colonies were observed at the concentration of 0.1563 µL/plate both in the absence and presence of metabolic activation system. No colonies were observed at the concentrations of 0.3125, 0.625, 1.25, 2.5 and 5.0 µL/plate both in the absence and presence of metabolic activation system. Normal growth was observed at the test concentrations of 0.0195 and 0.0098 µL/plate and negative control. Hence, 0.08 µL/plate of 3,4-dichloro-benzotrichloride was selected as the highest concentration to be tested in the mutagenicity test both in the absence and presence of metabolic activation system, for all the tester strains.

Mutagenicity Tests:
Trial I
Inhibition of background lawn and micro-colonies were observed at the concentration of 0.08 µL/plate in the tester strains of TA1537 and TA100 both in the absence and presence of metabolic activation system and in tester strains of TA1535, TA98 and TA102 in the absence of metabolic activation system. Therefore, concentration of 0.08 µL/plate was considered for statistical analysis, only for tester strains of TA1535, TA98 and TA102 in the presence of metabolic activation system.
A positive increase in the number of revertant colonies was not observed in any of the tester strains at any of the tested concentration when compared with the concurrent negative control. The results revealed that there was no positive mutagenic effect in strains TA1537, TA1535, TA98, TA100 and TA102 both in the absence and presence (5% v/v S9 mix) of metabolic activation system at any of the tested dose levels of 0.0025, 0.005, 0.01, 0.02, 0.04 and 0.08 µL/plate when compared with the concurrent negative control. Statistical analysis did not reveal any significant effects.

Trial II
Inhibition of background lawn and micro-colonies were observed at the concentration of 0.08 µL/plate in the tester strains TA1537 and TA100 both in the absence and presence of metabolic activation system and in the absence of metabolic activation system in tester strains TA1535, TA98 and TA102. Therefore, concentration of 0.08 µL/plate was considered for statistical analysis, only for tester strains of TA1535, TA98 and TA102 in the presence of metabolic activation system.
A positive increase in the number of revertant colonies was not observed in any of the tester strains both in the absence and presence (10% v/v S9 mix) of metabolic activation at any of the tested concentrations of 0.0008, 0.0020, 0.0051, 0.0128, 0.032 and 0.08 µL/plate when compared with the concurrent negative control. Results revealed that there was no positive mutagenic effect in strains TA1537, TA1535, TA98, TA100 and TA102 both in the absence and presence (10% v/v S9 mix) of metabolic activation at any of the tested concentrations when compared with the concurrent negative control. Statistical analysis did not reveal any significant effects.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mean Count of His⁺Revertant Colonies in NegativeControls, Positive Controls and Treatment Plates in the Absence ofMetabolic Activation (Trial I)

Concentration of 3,4-Dichloro-benzo-trichloride (µL/plate)	His^+Revertant Colonies/Plate (Absence of Metabolic Activation)
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	4.00	2.00	14.33	2.08	23.67	3.51	138.00	6.24	220.00	7.81
0.0025	4.33	1.15	15.33	1.53	23.67	2.52	136.67	4.16	219.67	5.51
0.005	4.33	1.53	13.33	1.53	23.00	1.00	133.00	4.36	213.67	3.21
0.01	4.67	0.58	12.33	1.15	24.00	1.73	135.33	11.50	224.67	4.04
0.02	3.67	0.58	12.00	1.00	22.33	1.53	133.67	2.52	223.00	3.00
0.04	4.67	0.58	9.00	1.00	17.67	2.08	115.00	7.94	221.67	5.51
0.08	-	-	-	-	-	-	-	-	-	-
PC	355.67	44.99	323.33	57.27	707.00	76.54	1256.67	102.79	1901.67	120.16
PC-2Aa	-	-	-	-	-	-	138.67	3.21	-	-

Key: SD = Standard Deviation, NC = Negative Control,DMSO = Dimethyl Sulphoxide,PC = Positive Control {TA1537 = 9-Aminoacridine (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C          (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100),- = Not applicable.

Mean Count of His⁺Revertant Colonies in Negative Controls, Positive Controls and Treatment Plates in the Presence ofMetabolic Activation(Trial I)

Concentration of 3,4-Dichloro-benzotrichloride(µL/plate)	His^+Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	5.33	1.53	18.67	3.79	30.67	7.37	150.67	3.51	235.67	15.04
0.0025	5.67	1.15	17.67	3.06	31.00	2.65	156.00	7.55	223.67	6.11
0.005	5.33	1.53	18.33	3.51	32.00	2.65	152.67	10.50	223.33	8.08
0.01	7.33	0.58	17.67	2.08	27.33	4.04	147.00	4.58	219.67	4.73
0.02	5.33	0.58	17.00	3.61	29.33	3.21	142.67	14.98	236.33	22.50
0.04	4.00	1.00	18.33	2.52	19.33	1.15	129.33	8.50	227.00	11.36
0.08	-	-	12.00	1.00	16.67	2.08	-	-	192.00	19.52
PC- 2Aa	304.33	25.50	315.67	60.54	332.33	28.59	1297.67	118.51	1817.67	170.90

Key:   SD =,= Negative Control,DMSO = Dimethyl Sulphoxide,PC = Positive Control, 2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5µg/plate for TA98 and TA100) and- = Not applicable.

Mean Count of His⁺Revertant Colonies in Negative Controls, Positive Controls

and Treatment Plates in the Absence ofMetabolic Activation (Trial II)

Concentration of 3,4-Dichloro-benzotrichloride(µL/plate)	His^+Revertant Colonies/Plate [Absence of Metabolic Activation]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	6.00	1.73	15.33	2.08	23.33	3.21	137.00	5.57	228.33	14.57
0.0008	6.33	1.53	18.67	3.79	23.33	5.86	139.33	4.16	221.67	5.86
0.0020	5.67	1.15	14.00	3.61	25.67	3.06	137.00	5.57	225.67	13.65
0.0051	4.33	1.15	15.00	1.00	23.67	5.03	135.33	2.08	221.00	6.56
0.0128	4.67	3.06	14.33	3.51	24.67	4.16	132.00	3.46	222.00	6.00
0.032	5.00	1.00	11.67	3.21	16.33	2.08	120.33	4.04	215.33	10.26
0.08	-	-	-	-	-	-	-	-	-	-
PC	320.67	29.09	392.00	20.00	630.33	48.06	1171.33	125.29	1933.00	69.22
PC-2Aa	-	-	-	-	-	-	139.67	3.06	-	-

Key: SD = Standard Deviation, NC = Negative Control,DMSO = Dimethyl Sulphoxide,PC = Positive Control {TA1537 = 9-Aminoacridine (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C          (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100) and- = Not applicable.

Mean Count of His⁺Revertant Colonies in Negative Controls, Positive Controls and Treatment Plates in the Presence ofMetabolic Activation(Trial II)

Concentration of 3,4-Dichloro-benzotrichloride(µL/plate)	His^+Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	7.67	3.06	20.33	3.51	24.00	3.00	154.00	10.15	235.33	15.95
0.0008	4.67	1.53	16.33	2.08	25.33	3.79	145.00	6.08	230.00	6.08
0.0020	8.33	3.06	18.33	2.52	22.67	5.03	143.00	10.00	216.33	12.66
0.0051	6.67	1.15	16.00	1.00	24.00	3.00	141.67	5.69	226.33	9.61
0.0128	5.00	2.00	18.33	2.08	23.67	1.53	146.33	13.65	230.67	5.03
0.032	7.33	0.58	14.00	2.00	17.33	2.08	126.33	4.73	221.33	6.81
0.08	-	-	11.00	2.65	14.67	1.53	-	-	188.67	17.47
PC	325.67	50.00	427.00	12.00	648.67	80.25	1203.00	52.74	1918.33	74.38

Key:    SD =,= Negative Control, DMSO = Dimethyl Sulphoxide,PC = Positive Control and 2Aa = 2-Aminoanthracene(10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100), - = Not applicable.

Applicant's summary and conclusion

Conclusions:

The negative control values in all the tester strains were within historical limits.
In both the trials, the positive controls exhibited a clear increase in number of revertants both in the absence and presence (5% and 10% v/v S9 mix) of metabolic activation with respective known mutagens when compared with the negative vehicle controls. This demonstrated the efficiency of the test system and suitability of the procedures employed in the study. The linear regression analysis did not reveal any significant increase in the number of revertant colonies in both the trials.

From the results of this study, it is concluded that 3,4-dichloro-benzotrichloride up to the concentration of 0.08 µL/plate both in the absence and presence (5% and 10% v/v S9 mix) of metabolic activation system, is non-mutagenic to all the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified conditions.

Executive summary:

This study was performed to evaluate 3,4-dichloro-benzotrichloride for its possible mutagenic activity, by the bacterial reverse mutation test, using five histidine deficient(his-)mutant tester strains of Salmonella typhimuriumviz., TA1537, TA1535, TA98, TA100 and TA102. The methods followed were as per the OECD N°471 (July, 1997).

The treatments were performed by the plate incorporation technique both in the absence and presence of metabolic activation (S9 mix). The S9 mix of 5% and 10% v/v consisted of an S9 fraction (Aroclor 1254 induced rat liver homogenate) supplemented with cofactors.

 Before conducting the mutagenicity test 3,4-dichloro-benzotrichloride was evaluated for its possible cytotoxicity in strain TA100, both in the absence and presence of S9 mix (5% v/v). Cytotoxicity to the tester strain was tested at the concentrations of 0.0098, 0.0195, 0.0391, 0.0781, 0.1563, 0.3125, 0.625,2.5 and 5.0 µL/plateboth in the absence and presence (5% v/vS9 mix) of the metabolic activation system. Cytotoxicity is characterised by inhibition of the background bacterial lawn and reduction in the number of colonies. Inhibition of background bacterial lawn was observed from concentrations of 0.1563to 5.0 µL/plate both in the absence and presence of metabolic activation system and at the concentration of 0.0781 µL/plate in the absence of metabolic activation system. Partial inhibitionof background bacterial lawnwasobserved at the concentration of 0.0391 µL/plate both in the absence and presence of metabolic activation system and at 0.0781µL/plate in thepresence of metabolic activation system. Reduction in the number of colonies was observed at the concentration of 0.0781µL/plate and micro-colonies were observed at the concentration of 0.1563 µL/plate both in the absence and presence of metabolic activation system. No colonies were observed from 0.3125 to 5.0 µL/plateboth in the absence and presence of metabolic activation system.Normal growth was observed at test concentrations of 0.0195 and 0.0098 µL/plate and negative control.

 Hence,µL/plateof3,4-dichloro-benzotrichloridewas selected as the highest concentration to be tested in the mutagenicity test both in the absence and presence of metabolic activation system, for all the tester strains.

Trial I

Based on the results of the cytotoxicity study,3,4-dichloro-benzotrichloridewas evaluated for its possible mutagenic effect in five strains ofSalmonella typhimuriumat thedose levels of 0.0025, 0.005,0.02,andµL/plate both in the absence and presence (5% v/v S9 mix) of metabolic activation system for trial I (factor 2). Triplicate plates were maintained for each test concentration of3,4-dichloro-benzotrichloride,negative and positive controls.

The results revealed that there was no positive mutagenic effect in strains TA1537, TA1535, TA98, TA100 and TA102 bothin the absence and presence (5% v/v S9 mix) of metabolic activation system at any of the tested dose levelswhen compared with the concurrent negative control.Statistical analysis did not reveal any significant effects.

Trial II

A second trial was conducted to confirm the negative results obtained in Trial I. In Trial II, the concentration spacing was modified using by factor 2.5 and the concentration of S9 mix was increased to 10% v/v.

The highest concentration beingµL/platefive lower concentrations viz., 0.032, 0.0128, 0.0051, 0.0020 and 0.0008µL/platewere tested both in the absence and presence (10% v/v S9 mix) of metabolic activation system.Triplicateplates were maintained for each test concentration of3,4-dichloro-benzotrichloride,negative and positive controls.

 

The results revealed that there was no positive mutagenic effect in strains TA1537, TA1535, TA98, TA100 and TA102 both in the absence and presence (10% v/v S9 mix) of metabolic activation system at any of the tested dose levels when compared with the concurrent negative control. Statistical analysis did not reveal any significant effects.

The values of negative control in all thetester strains during both the trials were within limits.

The positive controls exhibited a clear increase in the number of revertants both in the absence and presence of metabolic activation system(5% and 10% v/v S9 mix)with known mutagens when compared with the respective negative control. This demonstrated the efficiency of the test system and suitability of the procedures employed in the study.

From the results of this study, it is concluded that3,4-dichloro-benzotrichloride, up to the concentration of µL/plate both in the absence and presence(5% and 10% v/v S9 mix)of metabolic activation system, is non-mutagenic to all the fiveSalmonella typhimuriumtester strains viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified conditions.