Anilinoacetonitrile

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Principles of method if other than guideline:

The Ames II assay is a liquid version of the classical Ames test. The assay is performed in microwell plates using a modified fluctuation test protocol. Besides using the traditional tester strain TA 98 for identifiying mutages that induce small frameshift mutation in the his operon, a new set of 6 his- mutant strains, TA 7001-TA 7006 was developed to revert by unique base-pair substitutions.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Anilinoacetonitrile

EC Number:

221-129-0

EC Name:

Anilinoacetonitrile

Cas Number:

3009-97-0

Molecular formula:

C8H8N2

IUPAC Name:

2-(phenylamino)acetonitrile

Details on test material:

- Name of test material (as cited in study report): Phenylglycynnitril roh, ber. 100%
- Physical state: darkbrown liquid
- Purity test date: Mar 09, 1999
- Lot/batch No.: Mischprobe B 611 Lief. 20-24
- Storage condition of test material: room temperature

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium, other: 7001-7007

Metabolic activation:

with and without

Test concentrations with justification for top dose:

First experiment: 0, 4, 20, 100, 500, 2500, and 5000 µg/ml (TA98, TAMix)
Second experiment: 0, 4, 20, 100, 500, 2500, and 5000 µg/ml (TA98)
0, 1, 2, 4, 6, 8, 10 µg/ml (TAMix)

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Controls 2

Positive control substance:

other: mixture of 2-nitrofluorene and nitroquinoline-N-oxide

Remarks:

without S9 mix

Details on test system and experimental conditions:

The traditional tester strain TA 98 was selected by Ames and coworkers and is used for the detection of frameshift mutagens . The TA 7000 series of tester strains (TA 7001, TA 7002, TA 7003, TA 7004, TA 7005 and TA 7006) was constructed by Gee, P . et al. and have been designated "Ames II TM". These tester strains identifies the six possible base-pair substitution mutations; while TA 7001, TA 7002, and TA 7003 were developed to detect base changes at A : T base pairs, the strains TA 7004, TA 7005, and TA 7006 were constructed to detect base changes at G : C base pairs. Each of the base-specific strains carry a target missense mutation in the histidine biosynthetic operon that results in a reversion to prototrophy by base-substitution events unique to each strain. The strains of the TA 7000 series, each of which reverts by a specific base substitution, were used as a mixture, i .e. the so called "Mixed strains" (TA Mix) and consist of the strains TA 7001 - TA 7006 in equal proportions.
5 ml overnight cultures are mixed with 19 ml exposure medium, and were seeded in 24 well plates, where S9-Mix, if necessary and test substance was added. After 90 minutes of incubation 2.8 ml indicator medium was added into each well, and the suspension was plated into 48 wells of a 384 well plate (50 µl each). Each 48-well section of the 384-well plates is scored for the number of revertant wells (yellow). Individual plate counts, the mean number of revertant wells (yellow) per 48-well section and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. The number of positive wells out of a total of 48 wells is an indication of the frequency of reversion per replicate plate per dose and is compared to the number of spontaneous revertant wells obtained in the solvent control section . For a final evaluation the following comparisons/calculations are taken into consideration : An increase in the mean number of positive wells in dose groups compared to the mean values of the actual negative control (= 1 F). In cases of mean spontaneous mutation frequencies <1, the mean number is corrected to a level reflecting a mean = 1 in order for future calculation of fold increase. An increase in the mean of revertant wells in dose groups calculated on the basisof the baseline data of the actual experiment (= 2 F). The baseline is derived from the mean spontaneous revertant number plus 1 standard deviation from the distribution of spontaneous data. Separate baseline is derived from within each run against which data generated in that run are compared (= 3 F).
Generally, the experiment is to be considered valid if the following criteria are met : • The number of revertant wells in the negative controls was within the normal range of the historical control data for each tester strain. • The sterility controls revealed no indication of bacterial contamination . • The positive control articles both with and without S-9 mix induced a significant increase in the number of positive wells within the range of the historical control data.
The test chemical is considered positive in this assay if the following criteria are met : A dose-related and reproducible increase in the number of positive wells by a factor of • about 2 (calculated primarily on the basis of baseline data) in at least one tester strain either without S-9 mix or after adding a metabolizing system . A test substance is generally considered non-mutagenic in this test if : • The number of revertant wells for all tester strains were within the historical negative control range under all experimental conditions.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: 7001-7007

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table1: Results of the Ames II assay in the strains TA98 (a.without S9, b. with S9) and Mixed strains (c.without S9, d.with S9, e.without S9/Exp.2).

a.

Dose [µg/mL]	S9	RCSP 1	RCSP 2	RCSP 3	MEAN	MEANc	SD	1F	2F	3F
DMSO	-	2	2	0	1.3	1.3	0.94	1.0	0.6	0.5
4	-	1	3	2	2.0		0.82	1.5	0.9	0.7
20	-	1	5	1	2.3		1.89	1.8	1.0	0.8
100	-	1	1	1	1.0		0.0	0.8	0.4	0.4
500	-	1	1	0	0.7		0.47	0.5	0.3	0.2
2500	-	1	1	2	1.3		0.47	1.0	0.6	0.5
5000	-	1	1	0	0.7		0.47	0.5	0.3	0.2
4-NQO + 2 -NF	-	46	48	48	47.3		0.94	35.5	20.8	16.8

b.

Dose [µg/mL]	S9	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^c	SD	1F	2F	3F
DMSO	+	2	2	4	2.7	2.7	0.94	1.0	0.7	0.9
4	+	1	5	2	2.7		1.7	1.0	0.7	0.9
20	+	3	1	2	2.0		0.82	0.8	0.6	0.7
100	+	2	2	4	2.7		0.94	1.0	0.7	0.9
500	+	2	2	1	1.7		0.47	0.6	0.5	0.6
2500	+	1	3	1	1.7		0.94	0.6	0.5	0.6
5000	+	5	3	3	3.7		0.94	1.4	1.0	1.2
2 -AA	+	48	48	48	48.0		0.00	18.0	13.3	15.9

c.

Dose [µg/mL]	S9	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^c	SD	1F	2F	3F
DMSO	-	1	2	0	1.0	1.0	0.82	1.0	0.6	0.6
4	-	6	2	3	3.7		1.7	3.7	2.0	2.3
20	-	4	0	0	1.3		1.89	1.3	0.7	0.9
100	-	0	0	0	0.0		0.00	0.0	0.0	0.0
500	-	0	1	0	0.3		0.47	0.3	0.2	0.2
2500	-	2	0	1	1.0		0.82	1.0	0.6	0.6
5000	-	0	0	1	0.3		0.47	0.3	0.2	0.2
4-NQO + 2-NF	-	47	45	45	45.7		0.94	45.7	25.1	29.2

d.

Dose [µg/mL]	S9	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^c	SD	1F	2F	3F
DMSO	+	2	1	2	1.7	1.7	0.47	1.0	0.8	0.9
4	+	0	1	1	0.7		0.47	0.4	0.3	0.4
20	+	2	0	0	0.7		0.94	0.4	0.3	0.4
100	+	-	0	0	0.0		0.00	0.0	0.0	0.0
500	+	2	0	0	0.7		0.94	0.4	0.3	0.4
2500	+	1	0	0	0.3		0.47	0.2	0.2	0.2
5000	+	0	1	1	0.7		0.47	0.4	0.3	0.4
2-AA	+	44	46	44	44.7		0.94	26.8	20.9	23.7

e.

Dose [µg/mL]	S9	RCSP 1	RCSP 2	RCSP 3	MEAN	MEANc	SD	1F	2F	3F
DMSO	-	0	0	4	1.3	1.3	1.89	1.0	0.4	0.4
1	-	1	0	0	0.3		0.47	0.3	0.1	0.1
2	-	0	1	1	0.7		0.47	0.5	0.2	0.2
4	-	0	0	2	0.7		0.94	0.5	0.2	0.2
6	-	2	0	1	1.0		0.82	0.8	0.3	0.3
8	-	0	0	0	0.0		0.0	0.0	0.0	0.0
10	-	0	1	0	0.3		0.47	0.3	0.1	0.1
4-NQO + 2 -NF	-	47	46	46	46.3		0.47	34.8	14.4	14.9

OD                              optical density

REM                            remarks

B                                  Reduced backround growth

P                                  Precipitation

-                                   not tested

RCSP                          revertant colony selection plate (384-well)

SD                               standard deviation

MEAN                         mean of replicates

MEAN^c                        mean corrected: <1=1

F                                  factor

1F                                based on MEAN^c

2F                                baseline data / based on MEAN^c + SD

3F                                baseline data / based on MEAN + SD of run

4-NQO + 2-NF           4-Nitroquinoline: 0.0625 µg/mL + 2-nitrofluorene 0.25 µg/mL

2-AA                           2-Aminoanthracene: 5.0 µg/mL