Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2019 - 07 Jun 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich -Straße 7, 55116 Mainz
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibenzyldimethylammonium chloride
EC Number:
202-903-7
EC Name:
Dibenzyldimethylammonium chloride
Cas Number:
100-94-7
Molecular formula:
C16H20N.Cl
IUPAC Name:
dibenzyldimethylazanium chloride
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: solid, white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (protect against moisture, under N2).
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Storage stability: The stability of the test substance under storage conditions is guaranteed until 17 Jan 2020 as indicated by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: This study was performed in an aqueous test system. Due to the use of water (= ultrapure water) as vehicle the verification of the stability of the test substance in the vehicle is not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in water. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. All test substance formulations were prepared immediately before use.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : dissolved in water.

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:
Type and composition of metabolic activation system: S9 mix (supplemented with cofactors) derived from phenobarbital (i.p.) and β-naphthoflavone (orally) induced rat liver.
- source of S9 : prepared by the sponsor.
- method of preparation of S9 mix : At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied
by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
Test concentrations with justification for top dose:
0; 33; 100; 333; 1000; 2750 and 5500 μg/plate (SPT)
0; 33; 100; 333; 1000; 2750 and 5500 μg/plate (PIT)

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, due to the purity of the test substance 5.5 mg/plate was used as top dose in all experiments.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water.
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
other: not applicable
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene with S9 mix: 2.5 μg/plate for strains TA 1535, TA 100, TA 1537, TA 98. 60 μg/plate for strain: E. coli WP2 uvrA. 4-nitro-o-phenylenediamine without S9 mix: 10 μg/platefor strain: TA 98.
Remarks:
Sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at testing: approx. 10^9 cells per mL

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

DETERMINATION OF CYTOTOXICITY
- Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.
Evaluation criteria:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were 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 compatible with the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Precipitation: No precipitation of the test substance was found with and without S9 mix.

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle) historical control data: see table 1
- Positive historical control data: see table 2

Any other information on results incl. tables

Tab. 1: Historical Negative Controls

(based on Sorcerer Counter / Ames Study Mananger System (Sep 2017 - Sep 2018)

Strain

S9 Mix

Vehicle

No. of Plates

No. of Values

Min

Max

Mean

SD

 

 

 

 

 

 

 

 

 

TA 1535

Without

(All)

315

112

7

19

11

2.3

 

With

(All)

318

112

6

21

11

2.6

 

 

 

 

 

 

 

 

 

TA 100

Without

(All)

318

113

78

140

106

12.1

 

With

(All)

327

113

72

135

107

11.2

 

 

 

 

 

 

 

 

 

TA 1537

Without

(All)

309

112

4

12

8

1.7

 

With

(All)

309

112

5

13

9

1.6

 

 

 

 

 

 

 

 

 

TA 98

Without

(All)

318

113

12

27

18

2.9

 

With

(All)

312

113

18

45

25

4.0

 

 

 

 

 

 

 

 

 

E. coli

Without

(All)

306

113

19

38

26

4.2

 

With

(All)

306

113

18

40

26

4.1

Tab. 2: Historical Positive Controls

(based on Sorcerer Counter / Ames Study Mananger System (Sep 2017 - Sep 2018)

Strain

S9 Mix

Positive

control

No. of

Plates

No. of

Values

Min

Max

Mean

SD

 

 

 

 

 

 

 

 

 

TA 1535

Without

MNNG

252

88

2345

7317

4484

1042.2

 

With

2-AA

252

88

50

334

193

53.3

 

 

 

 

 

 

 

 

 

TA 100

Without

MNNG

252

88

814

5801

3443

1004.7

 

With

2-AA

258

88

413

2943

1563

492.8

 

 

 

 

 

 

 

 

 

TA 1537

Without

AAC

246

88

282

1497

809

237.1

 

With

2-AA

252

88

41

254

108

36.8

 

 

 

 

 

 

 

 

 

TA 98

Without

NOPD

252

88

484

1239

986

141.1

 

With

2-AA

249

88

161

2347

1098

379.9

 

 

 

 

 

 

 

 

 

E. coli

Without

4-NQO

246

88

176

1931

750

395.9

 

With

2-AA

246

88

66

259

115

45.7

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance 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.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA.

DOSE RANGE: 33 μg - 5500 μg/plate (SPT), 33 μg - 5500 μg/plate (PIT).

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was observed with and without S9 mix.

TOXICITY: A bacteriotoxic effect was occasionally observed depending on the strain and test conditions at and above 2750 μg/plate.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.