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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:
From 26 October 2017 to February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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

Constituent 1
Chemical structure
Reference substance name:
Quaternary ammonium compounds, dicoco alkyldimethyl, nitrites
EC Number:
275-532-1
EC Name:
Quaternary ammonium compounds, dicoco alkyldimethyl, nitrites
Cas Number:
71487-01-9
Molecular formula:
C18H40N2O2 - C42H88N2O2 (mainly [ca. 49%] C26H56N2O2)
IUPAC Name:
Quaternary Ammonium Compounds, Dicoco Alkyldimethyl nitrites
Test material form:
liquid: viscous

Method

Target gene:
Histidine (in S. Tryhimurium); Tryptophan (in E-coli)
Vehicle / solvent:
Anhydrous analytical grade dimethyl sulphoxide (DMSO).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
Test System
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, absence or presence of S-9 mix

Mutation Experiments
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites was tested for mutation (and toxicity) in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) in two separate experiments using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution

followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C and protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.
It should be noted that Mutation Experiment 2 data for strain TA98 in the absence of S-9 was obtained from a repeat experiment. In error, 2 concentrations (0.16 µg/plate and 1.6 µg/plate) were not treated in the initial treatments. Therefore, data from less than 5 analysable concentrations was obtained to make an assessment of mutagenicity. Data from the initial treatments are for reference only and included only for qualifying data for strain TA98 in the presence of S-9. This is because the data from the vehicle controls in the absence of S-9 are used to confirm the correct strain characteristics on that day of treatment for that experiment. Also, Mutation Experiment 2 data for strains TA1535 and TA1537 in the presence of S-9 were obtained from repeat experiments. The positive control treatments failed to generate an acceptable response in the initial and subsequent experiment for strain TA1537 treatments. The repeat treatments were performed using the same methodology as those used for the initial treatments and the data are presented as the Mutation Experiment 2 data for these strains.

Treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.
Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMSO) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response. Where mutation data from fewer than five treatment concentrations was obtained, an evaluation of the mutation data for the study as a whole was made.

Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or split in agar affected the accuracy of the automated counter.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:

1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98, TA100 and WP2 uvrA) or ≥3 fold (in strains TA1535 and TA1537) the concurrent vehicle control values
2. The positive trends/effects described above were reproducible.

The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if either of the above criteria were met.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

: Raw Plate Counts and Calculated Mutagenicity Data, Experiment 1, without S‑9
Strain Compound Conc. Level Mean Standard Deviation Fold Increase Revertant Numbers Per Plate
(µg/plate)
TA98 DMSO - 23.7 5.5 - 21, 20, 30
Quaternary ammonium compounds, di-C12-18- 5 21.3 5.5 0.9 25, 24, 15
alkyldimethyl, nitrites 16 23.7 7.5 1 15, 28, 28
50 - - - - T, - T, - T
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
2NF 5 656.7 44.9 27.7 708, 637, 625
             
TA100 DMSO - 116 11.1 - 126, 104, 118
Quaternary ammonium compounds, di-C12-18- 5 104.7 6.1 0.9 106, 110, 98
alkyldimethyl, nitrites 16 82.3 4.9 0.7 88 S, 80 S, 79 S
50 - - - - T, - T, - T
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
NaN3 2 475 35.9 4.1 460, 516, 449
             
TA1535 DMSO - 12.7 1.2 - 12, 14, 12
5 12.7 6.7 1 7, 11, 20
Quaternary ammonium compounds, di-C12-18- 16 12.3 2.5 1 10, 12, 15
alkyldimethyl, nitrites 50 - - - - T, - T, - T
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
NaN3 2 430.3 23.9 34 423, 457, 411
             
TA1537 DMSO - 13 6.6 - 20, 7, 12
Quaternary ammonium compounds, di-C12-18- 5 12.3 3.5 0.9 12, 16, 9
alkyldimethyl, nitrites 16 10.7 5 0.8 10, 16, 6
50 6.7 2.1 0.5 6, 5, 9
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAC 50 222.7 32.1 17.1 246, 186, 236
             
WP2uvrA DMSO - 23 3.5 - 21, 21, 27
Quaternary ammonium compounds, di-C12-18- 5 29.7 1.5 1.3 28, 30, 31
alkyldimethyl, nitrites 16 27.3 10 1.2 16, 35, 31
50 24.3 2.1 1.1 26, 22, 25
160 - - - - T, - T, - T
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
NQO 2 425.3 34.1 18.5 443, 386, 447
             

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 1, with S‑9
Strain Compound Conc. Level Mean Standard Deviation Fold Increase Revertant Numbers Per Plate
(µg/plate)
TA98 DMSO - 36 10.8 - 33, 48, 27
Quaternary ammonium compounds, di-C12-18- 5 38.3 2.3 1.1 41, 37, 37
alkyldimethyl, nitrites 16 31.3 2.9 0.9 33, 28, 33
50 35.3 7.6 1 27, 42, 37
160 29 3.6 0.8 33 S, 28 S, 26 S
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
B[a]P 10 324.7 39.8 9 334, 281, 359
             
TA100 DMSO - 106.7 9 - 116, 98, 106
Quaternary ammonium compounds, di-C12-18- 5 106.3 2.5 1 104, 109, 106
alkyldimethyl, nitrites 16 118 7.2 1.1 124, 120, 110
50 100.3 6.4 0.9 93, 105, 103
160 70 7.5 0.7 63 S, 69 S, 78 S
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAN 5 1209.3 135.1 11.3 1348, 1078, 1202
             
TA1535 DMSO - 24 6.1 - 28, 27, 17
Quaternary ammonium compounds, di-C12-18- 5 18 6.6 0.8 11, 19, 24
alkyldimethyl, nitrites 16 20.3 1.5 0.8 20, 22, 19
50 17.7 5.9 0.7 22, 20, 11
160 12 2.6 0.5 10 S, 15 S, 11 S
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAN 5 243.7 33.2 10.2 282, 225, 224
             
TA1537 DMSO - 9 2 - 11, 9, 7
Quaternary ammonium compounds, di-C12-18- 5 9.3 0.6 1 10, 9, 9
alkyldimethyl, nitrites 16 8.7 4.7 1 7, 14, 5
50 10.7 3.5 1.2 14, 7, 11
160 10.3 3.5 1.1 7 V, 10 V, 14 V
500 - - - - T, - T, - T
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAN 5 366 25 40.7 374, 338, 386
             
WP2uvrA DMSO - 28.3 1.5 - 27, 30, 28
Quaternary ammonium compounds, di-C12-18- 5 32.3 11.6 1.1 19, 40, 38
alkyldimethyl, nitrites 16 33 10.1 1.2 42, 22, 35
50 28.7 6.4 1 24, 36, 26
160 31.7 7.2 1.1 27, 40, 28
500 3.7 1.5 0.1 4 V, 2 V, 5 V
1600 - - - - T, - T, - T
5000 - - - - T, - T, - T
AAN 15 203.3 15.1 7.2 186, 214, 210
             

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, without S‑9
Strain Compound Conc. Level Mean Standard Deviation Fold Increase Revertant Numbers Per Plate
(µg/plate)
TA98 DMSO - 15.7 5 - 11, 15, 21
Quaternary ammonium compounds, di-C12-18- 0.05 22.7 5.5 1.4 19, 20, 29
alkyldimethyl, nitrites 0.16 - - - - U, - U, - U
0.5 18.3 1.2 1.2 19, 19, 17
1.6 - - - - U, - U, - U
5 25.3 5.7 1.6 27, 19, 30
16 23 2.6 1.5 24, 25, 20
50 - - - - T, - T, - T
2NF 5 1517.7 202.5 96.9 1520, 1314, 1719
             
TA100 DMSO - 92 18.1 - 94, 109, 73
Quaternary ammonium compounds, di-C12-18- 0.05 89.3 7.2 1 81, 94, 93
alkyldimethyl, nitrites 0.16 101.7 15 1.1 87, 117, 101
0.5 89 13.1 1 77, 87, 103
1.6 91 8.9 1 88, 84, 101
5 83.3 5.1 0.9 89, 82, 79
16 67 10.1 0.7 58, 78, 65
50 - - - - T, - T, - T
NaN3 2 887 13 9.6 880, 879, 902
             
TA1535 DMSO - 9.3 3.1 - 12, 10, 6
Quaternary ammonium compounds, di-C12-18- 0.05 6.7 2.9 0.7 5, 5, 10
alkyldimethyl, nitrites 0.16 12.3 4.2 1.3 9, 17, 11
0.5 9 6.6 1 15, 10, 2
1.6 7.3 1.5 0.8 7, 6, 9
5 9.7 2.5 1 7, 12, 10
16 10.3 4.5 1.1 10, 15, 6
50 - - - - T, - T, - T
NaN3 2 702 37.3 75.2 683, 745, 678
             
TA1537 DMSO - 15 6 - 9, 21, 15
Quaternary ammonium compounds, di-C12-18- 0.16 15.7 2.9 1 14, 19, 14
alkyldimethyl, nitrites 0.5 15.3 1.5 1 15, 14, 17
1.6 11.7 0.6 0.8 11, 12, 12
5 11.7 2.5 0.8 12, 14, 9
16 6.7 2.1 0.4 9, 5, 6
50 - - - - T, - T, - T
160 - - - - T, - T, - T
AAC 50 289.3 72.2 19.3 332, 330, 206
             
WP2uvrA DMSO - 21.7 5 - 21, 17, 27
Quaternary ammonium compounds, di-C12-18- 0.16 22.7 6 1 29, 22, 17
alkyldimethyl, nitrites 0.5 22 2.6 1 25, 21, 20
1.6 19 3.5 0.9 15, 21, 21
5 23.3 6 1.1 29, 17, 24
16 17.3 1.5 0.8 16, 17, 19
50 20 4.6 0.9 24, 15, 21
160 - - - - T, - T, - T
NQO 2 513.3 26 23.7 487, 539, 514
             

Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, with S‑9
Strain Compound Conc. Level Mean Standard Deviation Fold Increase Revertant Numbers Per Plate
(µg/plate)
TA98 DMSO - 43.7 4.9 - 38, 47, 46
Quaternary ammonium compounds, di-C12-18- 0.5 32 14.7 0.7 19, 48, 29
alkyldimethyl, nitrites 1.6 29.3 6.4 0.7 34, 32, 22
5 30.3 9.1 0.7 40, 22, 29
16 30.3 6.5 0.7 30, 24, 37
50 33 6.1 0.8 26, 36, 37
160 30.7 6.5 0.7 24, 37, 31
500 - - - - T, - T, - T
B[a]P 10 330 17.3 7.6 340, 310, 340
             
TA100 DMSO - 120.7 2.1 - 123, 119, 120
Quaternary ammonium compounds, di-C12-18- 0.5 88 4 0.7 84 M B, 88 M B, 92 M B
alkyldimethyl, nitrites 1.6 124 7.2 1 118, 122, 132
5 121.7 8.5 1 113 M, 122, 130
16 113.7 24.4 0.9 135, 87 M B, 119
50 113.7 31.1 0.9 139, 123, 79
160 101.7 8.4 0.8 92, 107, 106
500 - - - - T, - T, - T
AAN 5 1587 26.9 13.2 1556, 1604, 1601
             
TA1535 DMSO - 15 4.6 - 11, 14, 20
Quaternary ammonium compounds, di-C12-18- 0.5 13 2.6 0.9 16, 11, 12
alkyldimethyl, nitrites 1.6 11.3 2.3 0.8 10, 14, 10
5 17.3 4.6 1.2 20, 20, 12
16 12.7 7.6 0.8 6, 21, 11
50 19 5 1.3 19, 14, 24
160 13.7 1.5 0.9 12 S, 15 S, 14 S
500 - - - - T, - T, - T
  AAN 5 180.7 21.7 12 156, 197, 189
TA1537 DMSO - 11.7 2.1 - 10, 14, 11
Quaternary ammonium compounds, di-C12-18- 0.5 9.7 5.5 0.8 15, 4, 10
alkyldimethyl, nitrites 1.6 9.7 0.6 0.8 10, 10, 9
5 9 2.6 0.8 11, 10, 6
16 10.3 1.2 0.9 11, 11, 9
50 14.7 4 1.3 11, 14, 19
160 8 6.1 0.7 1 M P, 12 M P, 11 M P
500 0.7 1.2 0.1 0 M P, 0 M P, 2 M P
AAN 5 228.3 5.5 19.6 222, 232, 231
             
WP2uvrA DMSO - 31 2.6 - 29, 34, 30
Quaternary ammonium compounds, di-C12-18- 5 37.7 0.6 1.2 38, 37, 38
alkyldimethyl, nitrites 16 30.3 13.8 1 46, 20 M B, 25
50 29.7 3.2 1 26, 32, 31
160 25.7 0.6 0.8 26, 26, 25
500 10.7 5 0.3 10, 6, 16
1000 - - - - T, - T, - T
1600 - - - - T, - T, - T
AAN 15 240.7 43.7 7.8 219, 291, 212
         

Applicant's summary and conclusion

Conclusions:
Quaternary ammonium compounds, di-C12 -18 -alkyldimethyl, nitrites, CAS No. 71487 -01 -9, EC No. 275 -532 -1, did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a rat liver metabolic activation system (S-9) in the OECD 471, Bacterial Reverse Phase Mutation study : Negative.
Executive summary:

A study was performed to evaluate the mutagenic potential of the test material (Quaternary ammonium compounds, di-C12 -18 -alkyldimethyl, nitrites, CAS No. 71487 -01 -9, EC No. 275 -532 -1)

The study was conducted according to OECD guideline 471 and according to GLP.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

These test conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) and a toxic concentration, in the absence and in the presence of a rat liver metabolic activation system (S-9) in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA).

Under the conditions of the study it was concluded that Quaternary ammonium compounds, di-C12-18-alkyldimethyl, nitrites did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA).