Acetic acid, 2-cyano-, 1,1'-[2,2-bis[[(cyanoacetyl)oxy]methyl]-1,3-propanediyl] ester

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:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in accordance with GLP

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Remarks:

BASF AG, Department of Toxicology

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pentaerythritoltetracyanoacetic ester

EC Number:

605-830-2

Cas Number:

178671-69-7

Molecular formula:

C17 H16 N4 O8

IUPAC Name:

Pentaerythritoltetracyanoacetic ester

Method

Target gene:

his, trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

21.4 - 5350 µg/plate (SPT); 4.0 - 2500 µg/plate (PIT)

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice ofsolvent: Due to the insolubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see "details on test system"

Details on test system and experimental conditions:

METHOD OF APPLICATION: Standard plate test (SPT) and preincubation test (PIT)

STANDARD PLATE TEST (1st experiment):
Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies are counted.
Doses: 0; 21.4; 107; 535; 2675 and 5350 µg/plate with and without S9-Mix.

PREINCUBATION TEST (2nd experiment):
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 370C for 48 -72 hours in the dark, the bacterial colonies are counted
Doses: 0; 4; 20; 100; 500 and 2500 µg/plate with and without S9-Mix (dose selection is based on the findings of the 1st experiment).

CONTROLS:
- NEGATIVE CONTROLS:
Sterility control: Additional plates are treated with soft agar, S-9 mix, buffer, vehicle or the test substance but without the addition of tester strains
Vehicle control: The vehicle control with and without S-9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.

- POSITIVE CONTROLS:
With S-9 mix
2-aminoanthracene (2-AA) (SIGMA, A-1381)
- 2.5 µg/plate, dissolved in DMS0 for strains TA 1535, TA 100, TA 1537, TA 98
- 60 µg/plate, dissolved in DMSO for strain Escherichia coli WP2 uvrA

Without S-9 mix
N-methyl-N'-nitro-N-nitrosoguanidine (MN NG) (FLUKA, 68051)
- 5 µg/plate, dissolved in DMSO for strains TA 1535, TA 100

4-nitro-o-phenylendiamine (NOPD) (SIGMA, N-9504)
- 10 µg/plate, dissolved in DMSO for strain: TA 98

9-aminoacridine (AAC) (SIGMA, A-1 135)
- 100 µg/plate, dissolved in DMSO for strain TA 1537

4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141)
- 5 µg/plate, dissolved in DMSO for strain E. coli WP2 uvrA

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer
is recorded for all test groups both with and without S-9 mix in all experiments.

Evaluation criteria:

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 revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: from ca. 535 to 2675 ug/plate onward (SPT); from ca. 100 to 500 ug/plate onward (PIT)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 2500 µg/plate onward

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 535 µg - 2675 µg/plate onward. In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions from about 100 µg - 500 µg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

STANDARD PLATE TEST

	Colony count (mean of triplicates)
	TA 1535		TA 100		TA 1537		TA 98		WP2 uvrA
Dose (µg/plate)	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
DMSO	19	19	106	119	9	12	27	36	34	37
21.4	18	16	102	103	9	10	22	30	27	31
107	14	17	103	100	6	10	20	31	29	33
535	11	13	104	98	6	8	15	26	32	29
2675	11	8	103	98	6	5	15	22	26	26
5350	8	9	76	90	3	6	12	19	22	22
positive control	848	172	871	950	999	137	927	730	1095	323

positive controls see "details on test system"

PREINCUBATION TEST

	Colony count (mean of triplicates)
	TA 1535		TA 100		TA 1537		TA 98		WP2 uvrA
Dose (µg/plate)	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
DMSO	18	19	112	126	9	11	24	34	36	36
4	15	13	114	125	8	8	22	31	35	33
20	13	14	103	116	7	9	21	27	30	33
100	14	8	114	116	7	6	18	21	29	35
500	9	9	112	112	5	8	14	21	28	30
2500	7	8	86	103	4	4	10	17	25	28
positive control	1015	227	912	839	818	106	942	858	894	306

positive controls see "details on test system"

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen.

Executive summary:

The test article was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The following strains were tested: TA 1535, TA 100, TA 1537, TA 98 and E. col i WP2 uvrA. Two plating methods, the standard plate test and the preincubation test, each in the presence and absence of a metabolic activating system (rat liver S9 mix), were employed. The test substance was found to be precipitating at concentrations of 2500 µg/plate onward. A bacteriotoxic effect was observed under all test conditions. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Therefore, the test substance is not considered to be mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.