Triclocarban

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:

13-12-2018 - 21-12-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Triclocarban

EC Number:

202-924-1

EC Name:

Triclocarban

Cas Number:

101-20-2

Molecular formula:

C13H9Cl3N2O

IUPAC Name:

1-(4-chlorophenyl)-3-(3,4-dichlorophenyl)urea

Details on test material:

-Substance Name: triclocarban
-Molecular Formula: C13H9Cl3N2O
-Molecular weight: 315.58 g/mol
-SMILES: Clc2ccc(NC(=O)Nc1ccc(Cl)cc1)cc2Cl
- Substance type: organic
- Physical state: solid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0.0 (NC), 0.0 (VC), 0.004, 0.013, 0.040, 0.125 and 0.396 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose 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 control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Precipitation was checked as insolubility to assess precipitation in the final mixture under the actual test conditions and evident to the unaided eye. Test item dissolved in DMSO at 50 mg/mL concentration was checked for precipitation. Different amounts of formulated test item preparation (50 mg/mL) were added to overlay agar (top agar) in test tubes to give various test item concentration of (maximum 5 mg/plate) and plated on minimal glucose agar (MGA) plates. Precipitation was noticed at 5 mg/plate, 3.75 mg/plate and 2.5 mg/plate concentration which were assumed to interfere with the scoring. At treatment concentration 1.25 mg/plate slight precipitation was observed which was assumed to be non-interfering with the scoring. Therefore 1.25 mg/plate was selected as highest concentration for pre-experiment.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations 0.0 (NC), 0.0 (VC), 0.0003, 0.001, 0.004, 0.013, 0.040, 0.125, 0.396 and 1.25 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).

Toxicity of the test item results in a reduction in the number of spontaneous revertants or clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.000 – 1.25 mg/plate based on the solubility and precipitation test. In TA 98 and TA 100 there was no reduction in colony count but reduction in background lawn was observed in treated concentration 1.25 (T8) mg/plate and no reduction in colony count as well as in background lawn in treated concentrations 0.396 (T7) mg/plate – 0.0003 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.0 (NC), 0.0(VC), 0.004, 0.013, 0.040, 0.125 and 0.396 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9). The concentrations used in the experiment (pre-experiment, Trial-I, Trial-II) were placed with (√10) half log interval.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	106	20	113	20
	R2	110	19	116	18
	R3	109	18	121	21
VC (0.00)	R1	116	24	125	24
	R2	120	23	121	27
	R3	117	25	123	26
T1 (0.0003)	R1	102	20	110	20
	R2	101	20	105	21
	R3	108	21	116	18
T2 (0.001)	R1	110	18	119	16
	R2	104	22	120	22
	R3	102	17	117	20
T3 (0.004)	R1	106	21	119	19
	R2	112	23	115	22
	R3	110	17	118	21
T4 (0.013)	R1	112	16	119	19
	R2	116	24	125	23
	R3	116	25	118	25
T5 (0.040)	R1	104	21	122	23
	R2	117	22	120	25
	R3	110	24	117	21
T6 (0.125)	R1	110	20	120	25
	R2	115	18	123	23
	R3	114	22	122	22
T7 (0.396)	R1	116	24	117	20
	R2	106	23	122	24
	R3	115	18	124	21
T8 (1.25)	R1	119	21	123	27
	R2	118	18	120	26
	R3	115	17	123	24
PC	R1	1608	904	1600	1280
	R2	1592	912	1528	1248
	R3	1616	1008	1544	1328

NC          =    Negative control

VC          =  Vehicle Control

PC          =    Positive control

R             =    Replicate

T             =    Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	11	20	106	256
	R2	4	9	19	110	224
	R3	5	10	18	109	220
VC (0.00)	R1	8	15	24	116	256
	R2	5	16	23	120	260
	R3	8	14	25	117	272
T1 (0.004)	R1	6	12	21	106	232
	R2	5	11	23	112	236
	R3	5	9	17	110	246
T2 (0.013)	R1	7	10	16	112	240
	R2	5	13	24	116	234
	R3	5	11	25	116	238
T3 (0.040)	R1	6	13	21	104	242
	R2	7	11	22	117	236
	R3	5	12	24	110	250
T4 (0.125)	R1	7	14	20	110	256
	R2	6	15	18	115	238
	R3	6	13	22	114	252
T5 (0.396)	R1	8	14	24	116	240
	R2	6	13	23	106	250
	R3	6	12	18	115	262
PC	R1	180	1188	904	1608	1808
	R2	188	1240	912	1592	1760
	R3	156	1232	1008	1616	1860

 

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	11	20	113	250
	R2	5	11	18	116	232
	R3	4	10	21	121	220
VC (0.00)	R1	6	15	24	125	276
	R2	7	14	27	121	260
	R3	8	17	26	123	258
T1 (0.004)	R1	5	12	19	119	242
	R2	6	11	22	115	240
	R3	6	13	21	118	238
T2 (0.013)	R1	7	13	19	119	222
	R2	6	12	23	125	244
	R3	5	10	25	118	246
T3 (0.040)	R1	6	11	23	122	250
	R2	5	15	25	120	246
	R3	6	14	21	117	252
T4 (0.125)	R1	7	15	25	120	244
	R2	6	14	23	123	238
	R3	6	15	22	122	246
T5 (0.396)	R1	6	16	20	117	262
	R2	7	14	24	122	256
	R3	7	12	21	124	252
PC	R1	172	448	1280	1600	1344
	R2	180	420	1248	1528	1352
	R3	190	388	1328	1544	1312

 

NC= Negative Control,VC= Vehicle Control,T =Test concentrati388on (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                                2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                               Sodium azide [10μg/plate]: TA 1535, TA 100                                                                                                                                

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	4	10	20	110	232
	R2	5	12	17	112	224
	R3	5	13	20	113	240
VC (0.00)	R1	6	12	25	125	276
	R2	7	14	26	128	268
	R3	8	13	23	125	250
T1 (0.004)	R1	5	12	21	110	236
	R2	5	14	20	116	222
	R3	6	10	22	118	240
T2 (0.013)	R1	5	12	23	119	242
	R2	4	14	20	122	248
	R3	6	12	22	120	230
T3 (0.040)	R1	7	13	20	119	236
	R2	5	12	24	116	238
	R3	5	15	25	116	234
T4 (0.125)	R1	7	14	21	117	268
	R2	5	13	22	118	242
	R3	6	11	21	120	244
T5 (0.396)	R1	6	12	24	121	260
	R2	6	13	25	122	256
	R3	7	12	23	123	258
PC	R1	188	1120	956	1088	1680
	R2	200	1232	940	1272	1632
	R3	192	1256	912	1128	1568

 

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	12	22	115	242
	R2	5	11	17	107	230
	R3	5	13	20	109	244
VC (0.00)	R1	6	16	25	123	270
	R2	7	13	23	136	264
	R3	7	14	25	125	264
T1 (0.004)	R1	4	12	22	107	246
	R2	5	12	21	105	242
	R3	7	13	20	123	242
T2 (0.013)	R1	6	12	22	124	236
	R2	5	13	20	120	250
	R3	7	15	24	119	248
T3 (0.040)	R1	4	16	23	121	254
	R2	6	13	24	124	252
	R3	7	12	21	126	248
T4 (0.125)	R1	4	14	20	117	258
	R2	5	13	26	117	262
	R3	6	15	25	123	258
T5 (0.396)	R1	7	16	24	129	250
	R2	6	11	21	124	248
	R3	6	12	20	120	262
PC	R1	200	380	1280	1520	1440
	R2	186	432	1344	1472	1448
	R3	212	448	1360	1488	1520

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control                                                                                 2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                                 Sodium azide [10μg/plate]: TA 1535, TA 100,                                                                                                                                     

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]     Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 4 -   MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	5.33	1.53	10.00	1.00	19.00	1.00	108.33	2.08	233.33	19.73
VC (0.00)	7.00	1.73	15.00	1.00	24.00	1.00	117.67	2.08	262.67	8.33
T1 (0.004)	5.33	0.58	10.67	1.53	20.33	3.06	109.33	3.06	238.00	7.21
T2 (0.013)	5.67	1.15	11.33	1.53	21.67	4.93	114.67	2.31	237.33	3.06
T3 (0.040)	6.00	1.00	12.00	1.00	22.33	1.53	110.33	6.51	242.67	7.02
T4 (0.125)	6.33	0.58	14.00	1.00	20.00	2.00	113.00	2.65	248.67	9.45
T5 (0.396)	6.67	1.15	13.00	1.00	21.67	3.21	112.33	5.51	250.67	11.02
PC	174.67	16.65	1220.00	28.00	941.33	57.87	1605.33	12.22	1809.33	50.01

 

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	5.00	1.00	10.67	0.58	19.67	1.53	116.67	4.04	234.00	15.10
VC (0.00)	7.00	1.00	15.33	1.53	25.67	1.53	123.00	2.00	264.67	9.87
T1 (0.004)	5.67	0.58	12.00	1.00	20.67	1.53	117.33	2.08	240.00	2.00
T2 (0.013)	6.00	1.00	11.67	1.53	22.33	3.06	120.67	3.79	237.33	13.32
T3 (0.040)	5.67	0.58	13.33	2.08	23.00	2.00	119.67	2.52	249.33	3.06
T4 (0.125)	6.33	0.58	14.67	0.58	23.33	1.53	121.67	1.53	242.67	4.16
T5 (0.396)	6.67	0.58	14.00	2.00	21.67	2.08	121.00	3.61	256.67	5.03
PC	180.67	9.02	418.67	30.02	1285.33	40.27	1557.33	37.81	1336.00	21.17

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  

2-Aminoanthracene [10μg/plate]:TA 102                                            

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD
(TRIAL II)

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.67	0.58	11.67	1.53	19.00	1.73	111.67	1.53	232.00	8.00
VC (0.00)	7.00	1.00	13.00	1.00	24.67	1.53	126.00	1.73	264.67	13.32
T1 (0.004)	5.33	0.58	12.00	2.00	21.00	1.00	114.67	4.16	232.67	9.45
T2 (0.013)	5.00	1.00	12.67	1.15	21.67	1.53	120.33	1.53	240.00	9.17
T3 (0.040)	5.67	1.15	13.33	1.53	23.00	2.65	117.00	1.73	236.00	2.00
T4 (0.125)	6.00	1.00	12.67	1.53	21.33	0.58	118.33	1.53	251.33	14.47
T5 (0.396)	6.33	0.58	12.33	0.58	24.00	1.00	122.00	1.00	258.00	2.00
PC	193.33	6.11	1202.67	72.59	936.00	22.27	1162.67	96.77	1626.67	56.19

 

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	5.00	0.00	12.00	1.00	19.67	2.52	110.33	4.16	238.67	7.57
VC (0.00)	6.67	0.58	14.33	1.53	24.33	1.15	128.00	7.00	266.00	3.46
T1 (0.004)	5.33	1.53	12.33	0.58	21.00	1.00	111.67	9.87	243.33	2.31
T2 (0.013)	6.00	1.00	13.33	1.53	22.00	2.00	121.00	2.65	244.67	7.57
T3 (0.040)	5.67	1.53	13.67	2.08	22.67	1.53	123.67	2.52	251.33	3.06
T4 (0.125)	5.00	1.00	14.00	1.00	23.67	3.21	119.00	3.46	259.33	2.31
T5 (0.396)	6.33	0.58	13.00	2.65	21.67	2.08	124.33	4.51	253.33	7.57
PC	199.33	13.01	420.00	35.55	1328.00	42.33	1493.33	24.44	1469.33	44.06

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.0 (VC), 0.0003, 0.001, 0.004, 0.013, 0.040, 0.125, 0.396 and 1.25 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0 (VC), 0.004, 0.013, 0.040, 0.125 and 0.396 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methodsi.e.Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test item with the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.