α,α,α-trifluoro-p-toluidine

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:

: from 18-MAR-1986 to 13-MAY-1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No indication of purity of test substance (no analytical certificate).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

EU Method B.13/14 (Mutagenicity - reverse mutation test using bacteria) 19.9.84

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

α,α,α-trifluoro-p-toluidine

EC Number:

207-236-5

EC Name:

α,α,α-trifluoro-p-toluidine

Cas Number:

455-14-1

Molecular formula:

C7H6F3N

IUPAC Name:

4-(trifluoromethyl)aniline

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 (see table in other information)

Test concentrations with justification for top dose:

0.05; 0.1; 0.5; 1; 2 µl/plate

Vehicle / solvent:

- VEHICLE: DMSO (0.1 ml/ml)
> Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details on test system

Details on test system and experimental conditions:

- VEHICLE: DMSO (0.1 ml/ml)
> Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria
> Vehicle controls tested: no
- NEGATIVE CONTROL(S): DMSO (100 µl/plate)
- POSITIVE CONTROL(S)
2-nitrofluorene in TA 98 and TA 1538 (0.001 µl/plate) without metabolic activation
9-aminoacridine in TA 1537 (0.05 µl/plate) without metabolic activation
ethylmethanesulphonate in TA 1535 (10 µl/plate) without metabolic activation
methylmethanesulfonate in TA 100 (0.1 µl/plate) without metabolic activation
Amino-2 anthracene: TA 98, TA 100, TA 1535, TA 1537, TA 1538 (0.002 µl/plate) with metabolic activation
>Justification for choice of positive control(s): Standard mutagen products.

DETAILS ON TEST SYSTEM AND CONDITIONS:
*Preliminary experiment: served to identify toxicity. The strain TA 100 was exposed without activation metabolic system to following concentrations: 0.5, 1, 5, 10, 50 and 100 µl/plate

*First experiment: 3 replicates of S. Typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 with and without metabolic activation system were exposed to 0.05, 0.1, 0.5, 1 or 2 µl/plate of test substance. A preparation of 2.5 ml of agar +0.1 ml of bacteria culture and 0.5 ml of S9 mix (if required) was prepared extemporaly and added to 100 ml of substance in DMSO at adapted concentration. This mixture was agitated and spread out over a layer of Vogel and Bonner medium. The plates were incubated at 37°C for 48h.
Reading of results was done with electronic meter of colonies.

*Second experiment: same conditions than first experiment.
For both experiments, positive and negative controls for each strain tested with and without S9 mix were done.
Sterility controls were done on product solutions tested, DMSO and S9 mix.
Genetic strain control with and without metabolic activation system and without test substance were done.

- METHOD OF APPLICATION: in agar (plate incorporation)
- DURATION:
> Exposure duration: 48 hours
- Expression time (cells in growth medium): 16 hours at 37°C in nutritive culture to obtain plate phase (around 2.10E9 bacteria/ml)
- NUMBER OF REPLICATIONS: 3
OTHER: Electronic count.

Evaluation criteria:

Results were considered positive by the authors when the revertant numbers were equal or more than 2 fold the negative control. Quantitative method of mutagen power was applied by the statistic method of least square to evaluate mutagen power.

Statistics:

no quantitative evaluation by linear regression (method of least square). Mutagen power was expressed in reverse/µl of product.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA100, TA1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2 µl/plate for both strains

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: TA98, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2 µl/plate for TA 98 and TA 1538 with S9, and 1 µl/plate for TA 1538 without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2 µl/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In a preliminary study, the strain TA 100 was exposed without activation metabolic system to following concentrations: 0.5, 1, 5, 10, 50 and
100 µl/plate.
The concentration of 5 µl/plate was toxic, and concentrations for the main study were determined as follow: 0.05, 0.1, 0.5, 1 and 2 µl/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table of results: Number of revertants per plate

 

Strain		S9-mix	Concentration (µl/plate)
			0	0.05	0.1	0.5	1	2	Positive controls
TA 98	1^st study	-	27/32/27	23/28/42	28/18/28	28/29/33	36/50/ 36	6/3/3T	216/144
		+	36/27/34	32/28/41	38/40/37	51/41/64	52/62/ 47	27/27/27 T	1841/1796
	2^ndstudy	-	41/19/23	34/29/28	37/24/32	31/40/36	37/31/ 33	7/11/11T	264/259
		+	34/38/25	37/34/32	41/37/41	38/41/43	67/62/ 56	3/12/6T	1520/1449
TA 100	1^st study	-	86/86/81	180/143/ 176	222/208/ 215	427/518/ 497	870/772/ 809	182/45/0 T	302/316
		+	100/124/ 130	155/178/ 171	237/211/ 250	472/567/ 541	895/909/ 941	68/6/96 T	2441/3076
	2^ndstudy	-	104/87/77	130/114/ 130	170/178/ 200	409/413/ 394	551/507/ 483	111/192/ 25 T	343/338
		+	98/95/100	148/133/ 140	189/160/ 184	422/485/ 456	607/594/ 568	268/298/ 289 T	1233/1135
TA 1535	1^st study	-	6/12/11	21/32/40	41/52/65	122/155/ 147	179/182/ 169	15/9/2T	5989/6781
		+	7/11/11	34/54/41	54/49/67	195/182/ 158	224/222/ 204	122/157/ 112 T	158/149
	2^ndstudy	-	7/12/14	27/40/33	69/46/59	100/130/ 139	158/130/ 142	25/29/18 T	6275/6611
		+	12/15/15	25/25/25	46/41/24	143/138/ 127	143/174/ 160	156/176/174	136/176
TA 1537	1^st study	-	6/5/6	10/3/6	6/5/12	7/18/11	9/14/18	1/2/1T	614/577
		+	5/6/5	6/6/7	9/5/9	20/11/12	25/16/10	3/1/0T	183/152
	2^ndstudy	-	10/5/6	3/6/5	7/6/3	12/14/9	16/18/11	2/0/0 T	620/549
		+	2/3/11	3/1/10	10/2/2	9/14/10	11/12/10	0/2/3 T	179/187
TA 1538	1^st study	-	27/25/23	27/24/19	18/23/24	33/18/28	15/18/12 T	1/1/0T	167/158
		+	36/40/16	38/40/37	42/41/33	24/31/31	36/24/34	19/18/19 T	1069/883
	2^ndstudy	-	19/15/15	18/15/21	9/12/14	19/27/15	12/7/16	0/3/0 T	182/262
		+	20/19/25	18/27/24	20/27/24	27/19/20	25/28/21	2/6/5T	713/656

T: cytotoxicity

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with and without metabolic activation

- INTERPRETATION OF RESULTS:
Regarding results and applying actual criteria evaluation (the test item is considered as mutagen if an increase in revertants number exceeding the
threshold of twice for strains TA 98, TA 100, and thrice for strains TA 1535, TA 1537, TA 1538 as compared with solvent control), the test item
presented mutagenic activity with and without S9 mix in Salmonella Typhimurium TA 100 and TA 1535 (base substitution mutagen). A weak positive response in Salmonella Typhimurium TA 1537 (frameshift mutagen) and negative response for strains TA 98 and TA 1538 (frameshift mutagen)
were observed.
Reproductibility between first, second experiment and positive control results in accordance with positive criteria, validated the study.

Executive summary:

In a reverse gene mutation assay in bacteria (Hazleton, 1986), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to paratrifluoromethylaniline at concentrations of0.05,0.1, 0.5, 1 or 2 µl/plate in the presence and absence of mammalian metabolic activation.
Paratrifluoromethylaniline was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains.

In this study, the test item presented mutagenic activity with and without S9 -mix in Salmonella typhimurium TA100 and TA1535. A weak positive response in TA1537 and negative response for TA98 and TA1538.

Therefore, p-TFMA is considered as mutagenic for Salmonella typhimurium under the test conditions.
 
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.