2-[(2-nitrophenyl)amino]ethanol

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:

From 14 July 2004 to 19 August 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-nitrophenyl)amino]ethanol

EC Number:

225-555-8

EC Name:

2-[(2-nitrophenyl)amino]ethanol

Cas Number:

4926-55-0

Molecular formula:

C8H10N2O3

IUPAC Name:

2-[(2-nitrophenyl)amino]ethanol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot Code - 17
- Expiration date of the lot/batch: 11 December 2005
- Purity test date: 11 December 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from exposure to light and moisture
- Stability under test conditions: the test article was stable in DMSO at ≤ -20°C for at least 12 and 14 days at 0.052 and 513 mg/mL, respectively
- Solubility and stability of the test substance in the solvent/vehicle:The test article was insoluble in water from 10 to 50 mg/mL, the concentrations tested. The test article formed a soluble and clear solution in dimethyl sulfoxide (DMSO) at approximately 500 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The vehicle used to deliver the test item to the test system was dimethyl sulfoxide (DMSO, CAS No.
67-68-5), obtained from Fisher Scientific. The 100 mg/mL concentration was prepared by diluting
the test article in dimethyl sulfoxide. Serial dilutions were prepared by diluting in dimethyl
sulfoxide. Test article dilutions were prepared immediately before use and delivered to the test
system at room temperature under yellow light. Dosing solutions were adjusted to compensate for
the purity (99.9%) of the test article.

Method

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: Wp2 uvrA (pKM101)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice

Test concentrations with justification for top dose:

In the initial mutagenicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 1800 or at 5000 μg per plate. Based on the findings of the initial mutagenicity assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test article was insoluble in water from 10 to 50 mg/mL, the concentrations tested. The test article formed a soluble and clear solution in dimethyl sulfoxide (DMSO) at approximately 500 mg/mL, the maximum concentration tested. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): 10E9 per mL

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): 48 to 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): After incubation at 2-8°C for 48 to 72 hours

NUMBER OF REPLICATIONS: duplicate per conditions in the pre screen test and triplicate per condition for the Main Test

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Counted either entirely by automated colony counter or entirely by hand unless the assay was
the preliminary toxicity assay or the plate exhibited toxicity.

NUMBER OF CELLS EVALUATED: not specified


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; Bacteria background lawn
- Any supplementary information relevant to cytotoxicity: using a dissecting microscope.Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the table 1 below.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain. Data sets for tester strains TA1535 and TA1537 were 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. Data sets for tester strains TA98, TA100 and WP2 uvrA (pKM101) were 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.

Statistics:

No statistical analysis was specified in the report

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the initial mutagenicity assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg per plate. No precipitate was observed. Toxicity was observed beginning at 1800 or at 5000 μg per plate. Based on the findings of the initial mutagenicity assay, the maximum dose plated in the confirmatory
mutagenicity assay was 5000 μg per plate.
A positive mutagenic response was observed with tester strain TA98 (8.0-fold, maximum increase) in the presence of S9 activation. No positive mutagenic response was observed with any of the remaining tester strains in the presence of S9 activation and with any of the tester strains in the absence of S9 activation.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
See table 4 below

Any other information on results incl. tables

 

TABLE 2 : Summary of Results – Initial Toxicity-Mutation Assay Table
Test Article Id	: GTS03975
Study Number	: AA95CG.508.BTL	ExperimentNo : B1
AverageRevertantsPer Plate ± Standard Deviation LiverMicrosomes:	None
Dose (µg/plate)	TA98		TA100		TA1535		TA1537		WP2uvrA
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
Vehicle	24	0	136	15	28	1	8	1	247	4
2.5	20	3	139	17	22	9	2	1	224	6
7.5	14	7	111	7	18	5	10	4	242	22
25	15	6	130	8	17	0	2	1	226	29
75	17	1	106	9	23	3	5	0	219	9
200	14	6	137	5	17	11	6	1	196	16
600	16	6	118	2	28	4	7	1	149	3
1800	20	1	88	8	23	3	7	1	89	4
5000	0	0	0	0	0	0	0	0	0	0
Positive	403	44	482	65	379	51	753	297	946	74
LiverMicrosomes:	RatliverS9
Dose (µg/plate)	TA98		TA100		TA1535		TA1537		WP2uvrA
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
Vehicle	29	0	139	7	15	4	12	2	289	6
2.5	32	6	124	5	18	4	5	3	291	34
7.5	31	0	146	13	17	1	5	3	252	8
25	24	8	164	14	12	5	8	1	296	14
75	29	6	157	21	24	3	8	5	251	24
200	41	11	148	4	11	1	4	0	180	3
600	72	7	156	24	15	4	9	3	146	22
1800	233	11	154	21	14	2	17	3	87	8
5000	0	0	0	0	0	0	0	0	0	0
Positive	1249	96	891	73	97	4	122	16	1377	208
Vehicle = Vehicle Control
Positive = Positive Control (50 µL plating aliquot) Plating aliquot: 50 µL

 

 

 

TABLE 3 : Summary of Results – Confirmatory Mutagenicity Assay Table 22
Test Article Id	: GTS03975
Study Number	: AA95CG.508.BTL	ExperimentNo : B2
AverageRevertantsPer Plate ± StandardDeviationLiverMicrosomes:	None
Dose (µg/plate)	TA98		TA100		TA1535		TA1537		WP2uvrA
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
Vehicle	27	1	152	7	24	8	4	1	278	11
25	16	4	135	18	16	6	6	3	241	28
75	17	2	119	7	21	5	5	2	232	23
200	14	5	124	22	20	8	5	3	224	9
600	22	2	114	12	19	5	5	3	194	19
1200	18	4	135	6	21	7	4	1	180	15
1800	17	5	125	11	22	6	4	1	163	17
5000	0	0	0	0	0	0	0	0	0	0
Positive	456	36	529	56	429	16	1199	39	943	4
LiverMicrosomes:	RatliverS9
Dose (µg/plate)	TA98		TA100		TA1535		TA1537		WP2uvrA
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
Vehicle	39	8	150	19	21	2	7	3	268	11
25	35	3	154	32	16	3	11	5	284	30
75	38	6	122	19	20	3	8	2	295	13
200	37	4	171	9	17	1	9	3	268	6
600	57	9	135	10	17	1	9	3	255	13
1200	69	14	160	8	17	4	14	4	210	14
1800	169	82	152	13	16	6	10	2	183	9
5000	0	0	0	0	0	0	0	0	0	0
Positive	831	40	610	62	110	3	154	15	1420	70
Vehicle = Vehicle Control
Positive = Positive Control (50 µL plating aliquot) Plating aliquot: 50 µL

 

Table 4 :HistoricalData

Historical NegativeandPositive Control Values2001 – 2003   revertantsper plate
Strain	Control	Activation
		None				Rat Liver
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA98	Neg	15	5	5	49	20	7	5	49
	Pos	218	165	30	1981	695	385	40	2294
TA100	Neg	159	34	76	262	167	36	80	271
	Pos	606	140	271	2373	956	438	262	2922
TA1535	Neg	15	6	3	46	13	5	2	50
	Pos	344	140	16	1050	146	80	11	2246
TA1537	Neg	7	3	1	23	7	3	1	28
	Pos	639	386	13	2351	131	135	12	2021
WP2uvrA(pKM101)	Neg	189	34	131	297	198	39	132	301
	Pos	1169	395	470	2196	1186	326	380	1835
SD=standard deviation; Min=minimum value; Max=maximum value;Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone);Pos=positive control

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, test article GTS0375 was concluded to be positive in Salmonella typhimurium tester strain TA98 in the presence of Aroclor-induced rat liver S9.

Executive summary:

In this GLP-compliant study, The test item HC Yellow No. 2 was investigated for the induction of gene mutations in Salmonella typhimurium and Escherichia coli (Ames test) according to OECD 471 method.

The concentrations used in this study were :

initial experiment: 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg/plate without and with S9-mix

confirmatory experiment: 25, 75, 200, 600, 1200, 1800 and 5000 μg/plate without and with S9-mix

Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. The initial mutagenicity assay both without and with S9-mix was used to establish the dose range for the confirmatory assay and to provide a preliminary mutagenicity evaluation. The condition of the bacterial background lawn was evaluated for evidence of toxicity of the test item. Both experiments were performed without and with S9-mix using the preincubation method. Negative and positive controls were in accordance with the OECD guideline.

Results

In both the initial and the confirmatory experiment without and with S9-mix no precipitation occurred whereas toxicity was seen at the highest dose tested, 5000 μg/plate. A biologically relevant and more or less dose dependent increase in the number of revertant colonies was observed in strain TA98 after exposure to the test item in the presence of S9-mix. No mutagenic response was observed in the other tester strains in the presence of S9-

mix and with any of the tester strains in the absence of S9-mix.

Conclusion

Under the experimental conditions used the test item was mutagenic in this gene mutation tests in bacteria.