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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(21 Jul 1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Batch 20120706
Purity 83%
Other components: Manganese sulfate and sodium sulfate
Water 2.3%
Physical state: solid, beige
storage: in the refrigerator
Stability; guaranteed until Dec 31, 2018
Specific details on test material used for the study:
purity: 83% UVCB
physical state: solid, beige
Other components: water, inorganic salts

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
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
1st Experiment (Standard plate test with and without S9 mix): 0; 33; 100; 333; 1000; 3200 and 6400 μg/plate
2nd Experiment (Preincubation test with and without S9 mix): 0; 33; 100; 333; 1000; 3200 and 6400 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Positive controls:

With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA
Evaluation criteria:
Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment 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
Key result
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: A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 333 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward.

Any other information on results incl. tables

Table 1: Standard plate test without metabolic activation

Strain  Test group  Dose Mean revertants per plate Standard deviation Factor  Individual revertant colony counts
(μg/plate)
TA 1535 Acetone - 8.3 1.2 - 7, 9, 9
Test item 33 9.3 2.1 1.1 7, 10, 11
100 10.7 2.5 1.3 8, 11, 13
333 10.3 1.5 1.2 9 P, 10 P, 12 P
1000 7.3 0.6 0.9 8 P, 7 P, 7 P
3200 8.7 2.5 1.0 11 P, 9 P, 6 P
6400 8.0 2.6 1.0 7 P, 6 P, 11 P
MNNG 5.0 5389.3 255.2 646.7 5428, 5623, 5117
TA 100 Acetone - 89.3 13.7 - 83, 105, 80
Test item 33 101.3 5.7 1.1 106, 103, 95
100 101.0 14.7 1.1 104, 85, 114
333 96.0 7.8 1.1 92 P, 105 P, 91 P
1000 89.7 8.1 1.0 99 P, 86 P, 84 P
3200 94.3 1.2 1.1 95 P, 93 P, 95 P
6400 74.7 4.0 0.8 74 P, 71 P, 79 P
MNNG 5.0 3395.0 185.3 38.0 3504, 3181, 3500
TA 1537 Acetone - 5.0 1.0 - 6, 5, 4
Test item 33 3.7 1.2 0.7 3, 5, 3
100 5.7 0.6 1.1 6, 6, 5
333 5.3 2.1 1.1 3 P, 6 P, 7 P
1000 4.7 1.5 0.9 6 P, 5 P, 3 P
3200 3.3 0.6 0.7 3 P, 3 P, 4 P
6400 3.0 2.0 0.6 5 P, 1 P, 3 P
AAC 100 673.0 127.9 134.6 684, 540, 795
TA 98 Acetone - 17.7 6.5 - 18, 11, 24
Test item 33 17.7 0.6 1.0 17, 18, 18
100 16.7 1.5 0.9 15, 17, 18
333 20.7 2.5 1.2 21 P, 23 P, 18 P
1000 12.0 1.0 0.7 11 P, 13 P, 12 P
3200 12.3 1.5 0.7 11 P, 14 P, 12 P
6400 9.7 1.2 0.5 9 P, 9 P, 11 P
NOPD 10 1294.3 54.0 73.3 1326, 1325, 1232
E.coli Acetone - 25.3 1.5 - 27, 25, 24
Test item 33 26.7 1.5 1.1 25, 28, 27
100 24.3 4.6 1.0 27, 27, 19
333 24.0 1.7 0.9 25 P, 22 P, 25 P
1000 20.7 2.1 0.8 20 P, 23 P, 19 P
3200 21.0 7.2 0.8 23 P, 13 P, 27 P
6400 21.7 4.5 0.9 17 P, 22 P, 26 P
4-NQO 5 904.0 13.1 35.7 910, 913, 889

P = precipitation

Table 2: Standard plate test with metabolic activation

Strain  Test group  Dose Mean revertants per plate Standard deviation Factor  Individual revertant colony counts
TA 1535 Acetone - 6.3 1.2 - 5, 7, 7
Test item 33 6.0 1.7 0.9 5, 5, 8
100 7.7 2.3 1.2 9, 9, 5
333 5.7 2.3 0.9 7 P, 7 P, 3 P
1000 6.7 1.5 1.1 8 P, 7 P, 5 P
3200 5.3 1.5 0.8 7 P, 4 P, 5 P
6400 6.0 1.0 0.9 5 P, 6 P, 7 P
2-AA 2.5 296.0 25.2 46.7 267, 309, 312
TA 100 Acetone - 97.7 3.2 - 100, 99, 94
Test item 33 91.3 9.5 0.9 88, 102, 84
100 99.7 4.2 1.0 101, 103, 95
333 101.0 7.5 1.0 109 P, 100 P, 94 P
1000 111.7 11.6 1.1 106 P, 104 P, 125 P
3200 76.3 7.5 0.8 84 P, 69 P, 76 P
6400 78.3 11.0 0.8 71 P, 73 P, 91 P
2-AA 2.5 2603.7 206.1 26.7 2814, 2595, 2402
TA 1537 Acetone - 10.0 3.6 - 13, 11, 6
Test item 33 9.0 1.7 0.9 11, 8, 8
100 10.7 4.2 1.1 6, 14, 12
333 8.3 2.5 0.8 8 P, 11 P, 6 P
1000 8.3 3.5 0.8 8 P, 5 P, 12 P
3200 5.7 1.5 0.6 6 P, 4 P, 7 P
6400 3.3 0.6 0.3 3 P, 4 P, 3 P
2-AA 2.5 216.0 32.2 21.6 190, 252, 206
TA 98 Acetone - 24.0 5.6 - 29, 25, 18
Test item 33 27.7 2.9 1.2 31, 26, 26
100 27.7 4.7 1.2 24, 33, 26
333 21.0 7.5 0.9 14 P, 20 P, 29 P
1000 15.7 1.2 0.7 15 P, 17 P, 15 P
3200 12.3 3.2 0.5 10 P, 16 P, 11 P
6400 13.3 1.2 0.6 12 P, 14 P, 14 P
2-AA 2.5 1781.0 188.5 74.2 1615, 1742, 1986
E.coli Acetone - 27.3 4.5 - 32, 23, 27
Test item 33 28.7 2.9 1.0 27, 32, 27
100 25.0 5.3 0.9 19, 29, 27
333 24.0 7.0 0.9 21 P, 19 P, 32 P
1000 22.7 5.0 0.8 22 P, 18 P, 28 P
3200 19.7 3.5 0.7 20 P, 16 P, 23 P
6400 14.0 2.0 0.5 14 P, 12 P, 16 P
2-AA 60 93.3 21.1 3.4 71, 96, 113

P = Precipitation

Applicant's summary and conclusion

Conclusions:
The substance was not mutagenic in the Ames test.
Executive summary:

In a key bacterial reverse mutation assay, the test material (CAS# 9008-34-8; 83%) was tested on Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli strain WP2 at concentrations of 0; 33; 100; 333; 1000; 3200 and 6400 μg/plate in the presence and absence of metabolic activation (±S9).

The test material was not found to be mutagenic in the Ames test.