Amines, bis(C11-14-branched and linear alkyl), O,O-di-iso-Pr phosphorodithioates

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:

2011-12-06 - 2012-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study in compliance with GLP

Data source

Reference

Reference Type:

other: approved draft report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis-[N-C13-(branched and linear)-alykl]-ammonium O,O-dipropan-2-yl phosphorodithioate

EC Number:

700-768-3

Cas Number:

1285610-71-0

Molecular formula:

C26H56N.C6H14O2PS2

IUPAC Name:

Bis-[N-C13-(branched and linear)-alykl]-ammonium O,O-dipropan-2-yl phosphorodithioate

Details on test material:

- Physical state: Liquid, yellowish
- Analytical purity: 98.2 ± 0.4 g/100 g
- Storage condition of test material: Refrigerator

Method

Target gene:

his and 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:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

0.33 μg - 5 500 μg/plate (SPT, Salmonella strains)
33 μg - 5 500 μg/plate (SPT, E. coli)
0.33 μg - 100 μg/plate (PIT, Salmonella strains)
33 μg - 5 500 μg/plate (PIT, E. coli)

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see "details on test system"

Remarks:

with and without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: Standard plate test and Preincubation test

DURATION
- Preincubation period: 20 minutes (preincubation method only)
- Exposure duration: 48 – 72 hours (both methods)

NUMBER OF REPLICATIONS: three test plates per dose level and control

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

CONTROLS
With S9 mix
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO for strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO for strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO for strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD)
- 10 μg/plate, dissolved in DMSO for strain: TA 98
• 9-aminoacridine (AAC) (SIGMA, A-1135) 1)
- 100 μg/plate, dissolved in DMSO for strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO for strain: E. coli WP2 uvrA

Evaluation criteria:

The test substance 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 S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least 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:

cytotoxicity

Remarks:

cytotoxicty was observed from 33 µg/plate in salmonella strains, and at 5500 µg/plate in E.coli

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found at 5500 μg/plate with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A strong bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp- revertants, reduction in the titer) was observed in the standard plate test and in the preincubation assay depending on the strain and test conditions from about 33 μg/plate onward with the Salmonella strains and at 5500 μg/plate using the strain E. coli.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experimental Result:

Experiment 1 (standard plate test)

	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
vehicle control	11	14	74	82	6	6	17	24	39	47
33	9	12	73	87	3	4	14	18	37	43
100	9	10	65	75	1	1	6	12	31	43
333	0	0	0	0	0	0	0	0	36	41
1000	0	0	0	0	0	0	0	0	34	35
2750	0	0	0	0	0	0	0	0	30	31
5500	0	0	0	0	0	0	0	0	11	23
pos. control	802	156	836	718	395	137	587	616	892	257

Experiment 2 (standard plate test)

	TA 1535		TA 100		TA 1537		TA 98
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
vehicle control	14	15	97	107	6	11	22	27
0.33	15	15	96	117	7	10	22	33
1	15	16	93	100	6	11	22	29
3.3	13	17	86	115	8	9	24	35
10	12	17	87	99	6	11	23	31
33	15	15	77	107	5	9	21	26
100	8	11	9	47	1	3	6	13
pos. control	676	151	792	823	461	137	595	592

Experiment 3 (preincubation method)

	TA 1535		TA 100		TA 1537		TA 98		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
vehicle control	12	13	75	87	6	7	22	30
0.33	13	11	75	86	6	7	20	31
1	11	13	81	91	6	8	21	30
3.3	12	12	78	90	6	8	22	32
10	12	11	73	97	6	8	20	28
33	7	7	35	67	1	3	9	13	44	43
100									46	40
333									43	45
1000									35	40
2750									43	46
5500									33	41
pos. control	791	162	812	700	359	149	539	236	605	253

According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.